Mixtures and formulations comprising an alkyl ammonium EDTA salt

ABSTRACT

The present invention relates to mixtures comprising: i) at least one lipid and/or at least one oil; and ii) an alkyl ammonium EDTA salt; wherein the mixture has a water content in the range of 0 to 1.0 wt %. The invention further relates to mixtures which are pre-formulations, methods of treatment comprising administration of such pre-formulations, to pre-filled administration devices and kits containing the formulations, to the use of an alkylammonium EDTA salt to reduce the decomposition of the lipid components and/or any active agent contained within the pre-formulation, and to alkyl ammonium EDTA salts as described herein.

FIELD OF THE INVENTION

The present invention relates to mixtures comprising lipids and anantioxidant. The present invention also relates to formulationprecursors (pre-formulations) that upon exposure to water or aqueousmedia, such as body fluids, spontaneously undergo a phase transitionthereby forming a controlled release matrix. In particular, theinvention relates to mixtures, pre-formulations and compositions havingan improved resistance to oxidation.

BACKGROUND TO THE INVENTION

Many bioactive agents including pharmaceuticals, nutrients, vitamins andso forth have a “functional window”. That is to say that there is arange of concentrations over which these agents can be observed toprovide some biological effect. Where the concentration in theappropriate part of the body (e.g. locally or as demonstrated by serumconcentration) falls below a certain level, no beneficial effect can beattributed to the agent. Similarly, there is generally an upperconcentration level above which no further benefit is derived byincreasing the concentration. In some cases increasing the concentrationabove a particular level results in undesirable or even dangerouseffects.

Some bioactive agents have a long biological half-life and/or a widefunctional window and thus may be administered occasionally, maintaininga functional biological concentration over a substantial period of time(e.g. 6 hours to several days). In other cases the rate of clearance ishigh and/or the functional window is narrow and thus to maintain abiological concentration within this window regular (or even continuous)doses of a small amount are required. This can be particularly difficultwhere non-oral routes of administration (e.g. parenteral administration)are desirable or necessary, since self-administration may be difficultand thus cause inconvenience and/or poor compliance. In such cases itwould be advantageous for a single administration to provide activeagent at a therapeutic level over the whole period during which activityis needed.

Some patients undergoing treatment will typically require a therapeuticdose to be maintained for a considerable period and/or ongoing treatmentfor many months or years. Thus a depot system allowing loading andcontrolled release of a larger dose over a longer period would offer aconsiderable advantage over conventional delivery systems.

Certain of the formulations of the present invention generate anon-lamellar liquid crystalline phase following administration. The useof non-lamellar phase structures (such as liquid crystalline phases) inthe delivery of bioactive agents is now relatively well established. Amost effective lipid depot system is described in WO2005/117830, and ahighly preferred lipid depot is described in that document. However,there remains scope for achieving depot formulations having improvedperformance in several respects.

Lipid controlled-release delivery systems have been developed withactive agents including GLP-1 (WO2006/131730), somatostatin analogues(WO2006/075124), LHRH analogues (WO2006/075125), as well as non-peptidessuch as buprenorphine (WO2014/016428). Lipid systems are also of valuein treatment in their own right and need not include active agents. Forexample, the FDA approved oral liquid Episil® alleviates the pain causedby oral mucositis and other inflammatory conditions of the mouth byforming a lipid barrier in the oral cavity, but does not require anyactive agent.

A particularly versatile combination of lipids is glycerol dioleate(GDO) and phosphatidyl choline (PC). However, sustained releasedformulations can be produced with a wide variety of other lipidcomponents including tocopherol (WO2006/075123), derivatives of sorbitol(WO2016/102683), triglycerides (WO2016/066655), and a variety ofphospholipid components including phosphatidyl ethanolamines(WO2013/083459 and WO2013/083460).

Both the lipid components, particularly unsaturated lipids, and anyactive agent contained in the pre-formulation or sustained releasecomposition are susceptible to oxidation, either during storage or invivo. It is desirable to decrease the extent of oxidation sinceoxidation processes may reduce the content of active agent and/orcontribute to the formation of unwanted decomposition products. This inturn reduces the shelf life of a product.

One particular factor contributing to oxidation in lipid compositions isthe presence of trace amounts of metal ions, particularly transitionmetals such as iron (Fe). Even when the lipid components are of highpurity grade it is often difficult to entirely remove traces of suchions. It is thought that equipment used for the manufacture of lipidformulations commonly includes stainless steel which can leach smallamounts of metal ions (particularly Fe) into the mixture. It istherefore common to include an antioxidant in lipid formulations. Thesegenerally function by chelating any metal ions, thereby hindering theirparticipation in oxidation processes.

It is a prerequisite that any antioxidant must be soluble in the lipidmixture, e.g. pre-formulation. It is described in WO2012/160213 that acarefully controlled amount of water can be included in lipidpre-formulations without causing a phase change into a liquidcrystalline phase. In pre-formulations containing an appreciable aqueouscontent, it may be possible to include an effective amount of awater-soluble antioxidant such as ascorbic acid, inorganic salts ofmetal chelators, such as ethylenediaminetetraacetic acid (EDTA) (e.g.sodium or calcium salts) and citric acid. However, for certain activeagents it may be necessary to avoid prolonged exposure to water duringstorage (e.g. because the active agent is moisture sensitive), or a moredesirable release profile may be obtained without the inclusion of waterin the pre-formulation. The avoidance of water may also reduce theamount of trace metals which may be present, since metal ions aregenerally more soluble in water than in an organic solvent or lipidenvironment. In lipid formulations having a low water content it is notpossible to use conventional water-soluble antioxidants since these maynot have the requisite solubility in a substantially water-free lipidenvironment. It would therefore be advantageous to provide anantioxidant which is soluble in a substantially water-free lipidenvironment and which limits or prevents the oxidative degradation ofthe lipid components of the mixture, e.g. pre-formulation, and anyactive agent contained within. This is particularly the case for metalchelating agents such as EDTA where the standard inorganic salts (sodiumor calcium) are non-soluble or have negligible solubility in non-aqueousenvironments (e.g. lipid matrices).

WO2010/020794 describes thiolated antioxidants as offering particularadvantages in lipid systems and these are also suitable in non-aqueouslipid systems. However, for certain end uses the presence of a thiolatedantioxidant may not be acceptable.

This particularly applies, for example, to peptides or proteins havingthiolated groups or disulphide bridges. WO2010/020794 also mentions thepossibility of including EDTA or the sodium, disodium and calciumdisodium salts of EDTA as chelating agent although this is not an optionwhich is exemplified. The present inventors have established that EDTAor the common salts thereof are not soluble to any appreciable extent inthe types of lipid formulations described in WO2010/020794, i.e. thosebased on GDO, SPC and an organic solvent such as ethanol.

It has now surprisingly been established that effective amounts ofalkylammonium salts of EDTA can be dissolved in a non-aqueous lipidenvironment, and that the resulting mixtures, e.g. pre-formulations, arehighly resistant to oxidative decomposition during storage. Furthermore,although alkylammonium EDTA salts are believed to have an effect ondecreasing the decomposition by the expected mechanism of sequesteringmetal ions, the present invention may in some embodiments improveoxidation resistance above the level that can be accounted for solely bythis mechanism.

The inventors have established that the inclusion of alkylammonium EDTAsalts can prevent, or substantially decrease the rate of, oxidation of awide variety of lipid components and/or active agents contained therein.The inventors have found that the inclusion of alkylammonium EDTA cansubstantially reduce the loss of assay of active agent in drug samplestested in stability studies and thus increases shelf-life of the drugproduct. EDTA salts have the advantage that they are inexpensive, easilyproduced with a wide variety of countercations, and are generallyregarded as safe (and are widely used e.g. in pharmaceuticalapplications).

The stabilizing and shelf-life extending effect of alkylammonium EDTA asfound by the inventors may be not only related to the prevention orreduction of oxidation reactions but may be also related to theprevention or reduction of other chemical degradation reactions, e.g.hydrolysis, acylation, deamidation.

SUMMARY OF THE INVENTION

In a first aspect the invention provides a mixture of:

i) at least one lipid and/or at least one oil; and

ii) an alkyl ammonium EDTA salt (e.g. comprising an anion ofethylenediaminetetraacetic acid or an analogue thereof);

wherein the mixture has a water content in the range of 0 to 1.0 wt %.

In all aspects, ethylenediaminetetraacetic acid analogues and theircorresponding anions will typically be as described herein below.

The present invention also provides a pharmaceutical formulationcomprising an appropriate combination of lipid excipients, organicsolvent, and an alkylammonium EDTA salt, that can be used as adepot-precursor formulation (referred to herein for brevity as apre-formulation) to address one or more of the needs described above.

In a second aspect, the invention therefore provides a pre-formulationcomprising:

i) a lipid mixture comprising:

-   -   a) at least one of a mono-, di- or tri-acyl lipid and/or a        tocopherol;    -   b) optionally at least one phospholipid;    -   c) at least one biocompatible, organic solvent; and        ii) an alkyl ammonium EDTA salt (e.g. comprising an anion of        ethylenediaminetetraacetic acid or an analogue thereof); and        wherein the pre-formulation has a water content in the range of        0 to 1.0 wt %.

In a preferred embodiment the pre-formulation forms, or is capable offorming, at least one liquid crystalline phase structure upon contactwith excess aqueous fluid.

As used herein, the “lipid mixture” may be a “lipid controlled-releasematrix”.

A particularly preferred combination of components in some embodimentsis glycerol dioleate (GDO), phosphatidyl choline (PC), ethanol, andtetrakis(ethanolammonium) EDTA. The pre-formulation of all embodimentsmay further comprise an active agent, as described herein.

The pre-formulations are highly useful for the controlled and sustainedrelease of an active agent, especially those requiring or benefitingfrom a very flat release profile and/or minimal “burst” uponadministration. In a corresponding embodiment, the invention thereforeprovides for a mixture of:

i) a lipid mixture comprising:

-   -   a) at least one of a mono-, di- or tri-acyl lipid and/or a        tocopherol;    -   b) optionally at least one phospholipid;    -   c) at least one biocompatible, organic solvent;    -   d) an active agent; and        ii) an alkyl ammonium EDTA salt (e.g. comprising an anion of        ethylenediaminetetraacetic acid or an analogue thereof); and        wherein the mixture has a water content in the range of 0 to 1.0        wt %;

in the manufacture of a pre-formulation for use in the sustainedadministration of said active agent. In a preferred embodiment, thepre-formulation forms, or is capable of forming, at least one liquidcrystalline phase structure upon contact with excess aqueous fluid.

“Bioactive agents”, or “active agents” as referred to herein, may be anycompound having a desired biological or physiological effect, such as apeptide, protein, drug, antigen, nutrient, cosmetic, fragrance,flavouring, diagnostic, pharmaceutical, vitamin, or dietary agent andwill be formulated at a level sufficient to provide an in vivoconcentration at a functional level (including local concentrations fortopical compositions). In an embodiment the “active agent” is a naturalor synthetic peptide or non-peptide drug Active PharmaceuticalIngredient (API) which provides a therapeutic, palliative and/orprophylactic effect when administered to a suitable subject (typicallybeing one in need of such an effect).

In a further embodiment, the invention therefore provides a method forthe treatment of a human or non-human mammalian subject comprisingadministering to said subject a pre-formulation as described herein.Such a method may be for the treatment of a human or non-human mammaliansubject in need thereof to combat, (e.g. cure, improve, prevent orameliorate the symptoms of) at least one condition selected fromacromegaly, cancers, carcinomas, melanomas, tumours expressing at leastone somatostatin receptor, sst(2)-positive tumours, sst(5)-positivetumours, prostate cancers, gastro-entero-pancreatic endocrine tumours,gastro-entero-pancreatic neuroendocrine (GEP NE) tumours (GEP-NET), lungneuroendocrine tumours (lung NET), carcinoid tumours, insulinomas,TSH-secreting pituitary adenomas, gastrinomas, vasoactive intestinalpeptide (VIP) tumours and glucagonomas, elevated growth hormone (GH),elevated insulin-like growth factor I (IGF-I), varicial bleeding(especially espohageal), chemotherapy induced gastro intestinal problems(such as diarrhea), lymphorrhea, diabetic retinopathy, thyroid eyedisease, obesity, pancreatitis, and related conditions. Such methods areparticularly applicable where component d) is at least one somatostatinanalogue, as described herein. The preformulations as described hereinfor use in such methods form a further aspect of the invention.

Correspondingly, in a further aspect, the present invention provides theuse of a low viscosity mixture of:

i) a lipid mixture comprising:

-   -   a) at least one of a mono-, di- or tri-acyl lipid and/or a        tocopherol;    -   b) at least one phospho lipid;    -   c) at least one biocompatible, organic solvent; and        ii) an alkylammonium EDTA salt (e.g. comprising an anion of        ethylenediaminetetraacetic acid or an analogue thereof);        wherein the mixture has a water content in the range of 0 to 1.0        wt %; in the manufacture of a low viscosity pre-formulation        medicament for use in the in vivo formation of a depot for        treatment of at least one condition selected from acromegaly,        cancers, carcinomas, melanomas, tumours expressing at least one        somatostatin receptor, sst(2)-positive tumours, sst(5)-positive        tumours, prostate cancers, gastro-entero-pancreatic endocrine        tumours, gastro-entero-pancreatic neuroendocrine (GEP NE)        tumours, lung NE tumours (lung NET), carcinoid tumours,        insulinomas, gastrinomas, vasoactive intestinal peptide (VIP)        tumours and glucagonomas, TSH-secreting pituitary adenomas,        elevated growth hormone (GH), elevated insulin-like growth        factor I (IGF-I), varicial bleeding (especially espohageal),        chemotherapy induced gastro intestinal problems (such as        diarrhea), lymphorrhea, diabetic retinopathy, thyroid eye        disease, obesity, pancreatitis, and related conditions. Such        uses are particularly applicable where component d) is at least        one somatostatin analogue, as described herein.

Certain active agents (e.g. certain peptides) have benefits which arecosmetic rather than (or in addition to) therapeutic in nature. Sucheffects include weight-loss and/or hunger suppression as well as controlover skin or hair pigmentation, hair growth etc. The present inventiontherefore additionally provides a method of cosmetic treatment of ahuman or non-human mammalian subject comprising administering to saidsubject a pre-formulation as described herein. Such a cosmetic methodwill generally not be a method of therapy (i.e. will not havetherapeutic or medical benefit).

One of the advantages of the formulations of the present invention overmany other controlled-release compositions is that they are stable tostorage in their final form and thus little or no preparation isrequired at the time of administration. This allows the pre-formulationsto be ready-to-administer and also to be supplied in convenient,ready-to-administer form. In a further aspect, the invention thereforeprovides a pre-filled administration device containing a pre-formulationas described herein. Such a device will generally provide either asingle administration or multiple administrations of a composition whichwill deliver, for example, a dosage of active agent in the range of 1 μgto 15 mg/day, such as 0.1 mg to 15 mg/day or 1 μg to 5 mg/day.

In a further aspect the invention provides a kit comprising saidadministration device according to the invention.

The kit can optionally contain instructions for subcutaneous orintramuscular administration of said pre-formulation. Allpre-formulations described herein are suitable for use in such a kit andmay thus be contained therein.

The kits of the invention can optionally include additionaladministration components such as needles, swabs, and the like and willoptionally contain instructions for administration.

In a further aspect the invention provides an alkylammonium EDTA saltcomprising at least one alkyl ammonium cation of formula NR¹R²R³R^(4n+)as defined herein, with the proviso that the alkylammonium cation is nottrimethylammonium, tetramethylammonium, triethylammonium ortetraethylammonium.

BRIEF SUMMARY OF THE ATTACHED FIGURES

FIG. 1. Octreotide assay of Samples 53-54 as a function of time atstorage conditions 25° C./60% RH and 40° C./75% RH.

FIG. 2. Octreotide assay of Samples 55-60 as a function of EDTAconcentration (0-750 ppm or 0-0.075 wt %) at three time points (0, 1 and2 months) after storage at 40° C./75% RH.

FIG. 3. OCT assay in SPC/GDO/EtOH/PG based formulations in the absence(Sample 61) and presence of 100 ppm EDTA (Sample 62) as a function oftime at 25° C./60% RH. Formulations were stored in pre-filled glasssyringes.

FIG. 4. OCT assay in SPC/GDO/EtOH/PG based formulations as a function ofFe³⁺ concentration in the presence of 0, 25, 100 and 250 ppm EDTA(Samples 63-78) recorded at 1 month of storage at 40° C./75% RH.Formulations were stored in vials with ambient air in the headspace.

FIG. 5. OCT assay data in SPC/GDO/EtOH/PG formulations as a functionEDTA:Fe³⁺ molar ratio after 1 month of storage at 40° C./75% RH.Formulations were stored in vials with ambient air in the headspace.

FIG. 6. Assay (a) and Stability Index (b) values of OCT inSPC/GDO/EtOH/PG formulations as a function of time at 40° C./75%RH:without additives (Sample 79, reference), with EDTA(Na) (Sample 80),with EDTA(Na)/ETA (Sample 81), with EDTA (Sample 82), and with EDTA/ETA(Sample 83). Formulations were stored in vials with normal air in theheadspace. Except for the reference Sample 79, all formulations alsocontained 5 ppm Fe³⁺.

FIG. 7. OCT assay in SPC/GDO/EtOH/PG based formulations in the absence(Sample 79) and presence of 100 ppm EDTA solubilized in the lipidformulation by the use of ETA (Sample 84), DiETA (Sample 85) orethylenediamine (Sample 86) as a function of time at 40° C./75% RH.Formulations were stored in vials with normal air in the headspace.

FIG. 8. OCT assay in SPC/GDO/EtOH/PG (Sample 79—reference without EDTA,and Sample 84 with 100 ppm EDTA) based formulations as a function oftime at 40° C./75% RH. Formulations were stored in vials with normal airin the headspace.

FIG. 9. SOM assay in SPC/GDO/EtOH/PG (Sample 89—reference without EDTAand Sample 90 with 100 ppm EDTA) based formulations as a function oftime at 40° C./75% RH (a) and 25° C./60% RH (b). Formulations werestored in vials with normal air in the headspace.

FIG. 10. Assay (a) and Stability Index (b) values of GOS inSPC/GDO/EtOH/DMSO formulations without (Sample 93) and with 100 ppm EDTA(Sample 94) as a function of time at 40° C./75% RH. Both formulationscontained 5 ppm Fe³⁺ and were stored in vials with normal air in theheadspace.

FIG. 11. Assay (a) and Stability Index (b) values of O×Y inSPC/GDO/EtOH/DMSO formulations without (Sample 95) and with 100 ppm EDTA(Sample 96) as a function of time at 40° C./75% RH. Both formulationscontained 5 ppm Fe³⁺ and were stored in vials with normal air in theheadspace.

FIG. 12. Assay (a) and Stability Index (b) values of GRN inSPC/GDO/EtOH/DMSO formulations without (Sample 97) and with 100 ppm EDTA(Sample 98) as a function of time at 40° C./75% RH. Both formulationscontained 5 ppm Fe³⁺ and were stored in vials with normal air in theheadspace.

FIG. 13. Assay (a) and Stability Index (b) values of GOS inSPC/GMO/EtOH/DMSO formulations without (Sample 99) and with 100 ppm EDTA(Sample 100) as a function of time at 40° C./75% RH. Both formulationscontained 5 ppm Fe³⁺ and were stored in vials with normal air in theheadspace.

FIG. 14. Assay (a) and Stability Index (b) values of GOS inSPC/SbOil/EtOH/DMSO formulations without (Sample 101) and with 100 ppmEDTA (Sample 102) as a function of time at 40° C./75% RH. Bothformulations contained 5 ppm Fe³⁺ and were stored in vials with normalair in the headspace.

FIG. 15. Vial headspace oxygen concentration for SPC/GDO (50/50 w/w)based formulations without (Samples 103 and 104) and with 100 ppm EDTA(105 and 106) in the absence (a) and presence of 5 ppm Fe³⁺ (b) as afunction of time at 60° C./ambient RH. Formulations were stored in vialswith normal air in the headspace.

FIG. 16. Vial headspace oxygen concentration for SPC/GDO (35/65 w/w)based formulations without (Samples 107 and 108) and with 100 ppm EDTA(109 and 110) in the absence (a) and presence of 5 ppm Fe³⁺ (b) as afunction of time at 60° C./ambient RH. Formulations were stored in vialswith normal air in the headspace.

FIG. 17. Vial headspace oxygen concentration for SPC/GDO (50/50 w/w)based formulations without (Samples 103 and 104) and with 100 ppm EDTA(105 and 106) in the absence (a) and presence of 5 ppm Fe³⁺ (b) as afunction of time at 40° C./75% RH. Formulations were stored in vialswith normal air in the headspace.

FIG. 18. Vial headspace oxygen concentration for SPC/GDO (35/65 w/w)based formulations without (Samples 107 and 108) and with 100 ppm EDTA(109 and 110) in the absence (a) and presence of 5 ppm Fe³⁺ (b) as afunction of time at 40° C./75% RH. Formulations were stored in vialswith normal air in the headspace.

DETAILED DESCRIPTION OF THE INVENTION

Lipids and oils, particularly those having unsaturated groups, are proneto oxidation. Mixtures which comprise lipids or oils may thereforegradually decrease in purity over time e.g. during storage or use. Thisis undesirable and may lead to unwanted changes in the physical and/orchemical properties of the mixture. It is particularly important tominimise the amount of breakdown products in mixtures having apharmaceutical use, since breakdown products may be harmful to a patientand in any case often have to be kept within tightly controlled limits.

Lipids and oils are poorly miscible with water and so the water contentof lipids and oils is generally low. It is therefore difficult toformulate lipids or oils with water-soluble antioxidants. It wouldtherefore be desirable to find an antioxidant which could beincorporated with lipids or oils in order to prevent oxidation of themixture. The present invention addresses these problems.

The mixtures of the present invention are substantially non-aqueous andinclude at least one lipid and/or oil (component i) and at least onealkylammonium EDTA salt (component ii). In a preferred aspect, themixture is a pre-formulation. The pre-formulations of the presentinvention are lipid-based, are substantially non-aqueous and form adepot composition upon contact with an aqueous fluid. As used herein,the terms “formulation” or “pre-formulation” relate to the mixture ofcomponents (i) and (ii) (component (i) comprising components (a), (c),and optionally (b) and (d)), which is typically of low viscosity. Theterm “depot” relates to the composition which is formed upon exposure ofthe pre-formulation to excess aqueous fluid, e.g. as occurs duringnumerous parenteral administration routes. Without wishing to be boundby theory, it is thought that this change is brought about at least inpart by exchange of solvent (c) for aqueous fluid. The depot typicallyhas a much higher viscosity than the corresponding pre-formulation andprovides for the gradual release of any active agent contained withinthe depot.

In a preferred aspect, the formulations of the present inventiongenerate a non-lamellar phase (e.g. non-lamellar liquid crystallinephase) following administration. The use of non-lamellar phasestructures (such as liquid crystalline phases) in the delivery ofbioactive agents is now relatively well established. A most effectivelipid depot system is described in WO2005/117830, and a suitable lipidmatrix for use in the present invention is described in that document,the full disclosure of which is hereby incorporated herein by reference.For a description of the most favourable phase structures of suchformulations, attention is drawn to the discussion in WO2005/117830 andparticularly to page 29 thereof. Preferably the pre-formulationaccording to the invention has an L₂ phase (liquid phase) structure oris a liquid solution or molecular solution.

All % are specified by weight herein throughout, unless otherwiseindicated. Percent (%) by weight may be abbreviated e.g. as wt %.Furthermore, the % by weight indicated is the % of the totalpre-formulation including all of the components indicated herein, unlessotherwise indicated. Where a percentage by weight is given in relationto component (d) the weight relates to the amount of free base (e.g.where a salt is used), unless otherwise indicated. In certain Examples,the wt % of a specified salt is provided but is indicated whereappropriate and may be readily converted to the corresponding weight offree base.

The pre-formulations can optionally consist of essentially only thecomponents indicated herein (including where appropriate additionaloptional components indicated herein below and in the attached claims)and in one aspect consist entirely of such components.

The lipid-based pre-formulations described herein comprise lipid mixture(i) which includes lipid components (a) an organic solvent (c), andoptionally (b) and (d), and an alkylammonium EDTA salt (ii).

The present inventors have now surprisingly established that byappropriate choice of antioxidant, the oxidation resistance of the lipidand/or oil, and in the case of pre-formulations, any active agentcontained in the pre-formulation, can be significantly improved.

Whilst various alkylammonium EDTA salts are known, for instance fromScott and Kyffin (Biochem. J. (1978) 169, 697-701), their use as anantioxidant in lipid systems and compatibility with such formulationshas been hitherto unknown. Scott and Kyffin describe the use of solubleEDTA salts in the demineralisation of bone samples, where EDTA acts as asequestering agent. A particularly suitable solution is said to be 80%aqueous ethanol containing 0.2 M trimethylammonium EDTA. No use in lipidformulations nor solubility in lipids is suggested. The purpose of theEDTA salt in the present invention is as a preservative or stabilityenhancing agent in lipid formulations, and is very different from thatdescribed previously.

Component i)—Lipid and/or Oil

In all embodiments of the invention the mixture comprises at least onelipid and/or oil (component i) and has a water content of 0-1.0 wt %.Mixtures of lipids, mixtures of oils, or mixtures of both lipids andoils may be used as component i).

As used herein, the term “oil” refers to saturated or unsaturated C5-C70hydrocarbons which are liquid at room temperature and pressure.Preferred oils for use in the invention are saturated or unsaturatedC10-C60 hydrocarbons, preferably saturated or unsaturated C10-C40hydrocarbons.

In an embodiment component i) is an oil which is suitable for use alubricant. Such oils will typically be saturated C10-C40 hydrocarbons.It is desirable that lubricants are resistant to oxidation, becauseoxidation tends to increase the viscosity of the lubricant.

In an embodiment component i) comprises, consists essentially of, orconsists of at least one fatty acid or fatty acid ester (lipid). Fattyacids/lipids differ from “oils” in that they contain a polar carboxylicacid or ester “head group” with the hydrocarbon chain forming anon-polar “tail” group. Fatty acid esters are esterified fatty acids.Fatty acids or esters used in the present invention may be solid orliquid at room temperature and pressure, preferably liquid.

Examples of non-polar “tail” groups include C₆-C₃₂ alkyl and alkenylgroups, which are typically present as long chain carboxylic acids orthe esters thereof. These are often described by reference to the numberof carbon atoms and the number of unsaturations in the carbon chain.Thus, CX:Z indicates a hydrocarbon chain having X carbon atoms and Zunsaturations. Examples particularly include lauroyl (C12:0), myristoyl(C14:0), palmitoyl (C16:0), phytanoyl (C16:0), palmitoleoyl (C16:1),stearoyl (C18:0), oleoyl (C18:1), elaidoyl (C18:1), linoleoyl (C18:2),linolenoyl (C18:3), arachidonoyl (C20:4), behenoyl (C22:0) andlignoceroyl (C24:9) groups. For the avoidance of doubt, when referenceis made herein to the number of carbon atoms in the “chain” or “tail”this number includes the carbon atom of the —C(O)O— moiety, as isconventional in the art.

Thus, typical non-polar chains are based on the fatty acids of naturalester lipids, including caproic, caprylic, capric, lauric, myristic,palmitic, phytanic, palmitolic, stearic, oleic, elaidic, linoleic,linolenic, arachidonic, behenic or lignoceric acids, or thecorresponding alcohols. Preferable non-polar chains are palmitic,stearic, oleic and linoleic acids, particularly oleic acid.

The lipid(s) may be saturated or unsaturated, but preferably comprise atleast 1 wt % unsaturated lipid (based on the total lipid content), suchas at least 5 wt % (5-100%), at least 15 wt % (15-100%), at least 30 wt% (30-100%), at least 50 wt % (50-100%) or at least 80 wt % (80-100%).

In an embodiment component i) is a single fatty acid/fatty acid ester ormixture of fatty acids/fatty acid esters. Typically component i) willcomprise a mixture of saturated and unsaturated fatty acids. In apreferred embodiment the lipid(s) and/or oil(s) are extracted from anatural source.

In an embodiment component i) is an edible lipid such as almond oil,avocado oil, butter, canola oil, castor oil, coconut oil, corn oil,cottonseed oil, flaxseed oil, ghee, lard, linseed oil, macadamia oil,margarine, mustard oil, olive oil, palm oil, peanut oil, pumpkin seedoil, rice bran oil, safflower oil, sesame oil, soybean oil, sunfloweroil, tea seed oil, vegetable oil or walnut oil. For the avoidance ofdoubt, the above edible lipids are “lipids” rather than “oils” in thesense of component (i) because they contain fatty acids, particularly inthe form of fatty acid esters, rather than hydrocarbons.

In an embodiment component i) is as defined for component a) or b) asdescribed in subsequent sections.

In a particularly preferred embodiment the mixture consists essentiallyof, or consists of, components i) and ii).

Pre-Formulations

A pre-formulation is a subcategory of the “mixtures” describe above inwhich component i) is a lipid mixture and comprises at least one neutrallipid “component a)” and optionally at least one phospholipid “componentb)”. Pre-formulations additionally comprise component c) and optionallycomponent d) as described below.

Component a)—Neutral Lipid

Preferable ranges for component a) are 20-90 wt. % of thepre-formulation, preferably 30-70 wt. %, more preferably 33-60% (e.g.43-60%), particularly 38 to 43%.

Component “a” as indicated herein is at least one mono-, di- or triacyllipid comprising a polar “head” group and at least one non-polar “tail”group. Alternatively, component a) may comprise or consist oftocopherol(s). In a preferred aspect component a) comprises at least oneneutral di-acyl lipid (having no net charge at physiological pH).

As used herein, the term “acyl lipid” relates to a lipid componentcontaining a polyol “head” group and one or more apolar “tail groups”.In certain embodiments the polyol may be glycerol, a sugar or a hexitansuch as sorbitan. The term “hexitan” denotes a hexitol of formulaHOCH₂(CHOH)₄CH₂OH which has cyclised by formally losing one equivalentof water, to form a five or six membered ring, preferably a fivemembered furanose ring. Sorbitan is a particularly suitable “headgroup”, particularly as a component of a mono-acyl lipid component incertain embodiments.

In the case of di- and triacyl lipids, it is most preferred that thelipid component comprises a glycerol head group with two or three apolartail groups. The two or three non-polar groups may have the same or adiffering number of carbon atoms and may each independently be saturatedor unsaturated. Examples of non-polar groups include C₆-C₃₂ alkyl andalkenyl groups, which are typically present as the esters of long chaincarboxylic acids. These are often described by reference to the numberof carbon atoms and the number of unsaturations in the carbon chain.Thus, CX:Z indicates a hydrocarbon chain having X carbon atoms and Zunsaturations. Examples particularly include lauroyl (C12:0), myristoyl(C14:0), palmitoyl (C16:0), phytanoyl (C16:0), palmitoleoyl (C16:1),stearoyl (C18:0), oleoyl (C18:1), elaidoyl (C18:1), linoleoyl (C18:2),linolenoyl (C18:3), arachidonoyl (C20:4), behenoyl (C22:0) andlignoceroyl (C24:9) groups. Thus, typical non-polar chains are based onthe fatty acids of natural ester lipids, including caproic, caprylic,capric, lauric, myristic, palmitic, phytanic, palmitolic, stearic,oleic, elaidic, linoleic, linolenic, arachidonic, behenic or lignocericacids, or the corresponding alcohols. Preferable non-polar chains arepalmitic, stearic, oleic and linoleic acids, particularly oleic acid.

Mixtures of any number of monoacyl, diacyl and/or triacyl lipids may beused as component a). Preferably this component will include at least aportion of C18 lipids (e.g. a diacyl glycerol (DAG) having one or moreC18:0, C18:1, C18:2 or C18:3 non-polar groups), such as glyceroldioleate (GDO) and/or glycerol dilinoleate (GDL). A highly preferredexample is DAG comprising at least 50%, preferably at least 80% and evencomprising substantially 100% GDO.

Since GDO and other diacyl glycerols may be derived from naturalsources, there is generally a certain proportion of “contaminant” lipidhaving other chain lengths etc. In this context, “pure” GDO is adi-ester of glycerol and two C18:1 fatty acids. Any other diacylglycerol is considered to be an impurity. In one aspect, GDO as usedherein is thus used to indicate any commercial grade of GDO withconcomitant impurities (i.e. GDO of commercial purity). These impuritiesmay be separated and removed by purification but providing the grade isconsistent this is rarely necessary. If necessary, however, “GDO” may beessentially chemically pure GDO, such as at least 70% pure, preferablyat least 75% pure and more preferably at least 80% pure GDO.Correspondingly, the C18:1 content of GDO referred to herein may bearound 80%, preferably at least 85% and more preferably at least 90%.

It will be appreciated that any material used, including component a),may potentially include unavoidable trace impurities of metals,optionally including heavy metals. According to the certificates ofanalysis for commercially available GDO (e.g. from Croda), a typicalmaximum concentration of heavy metals (or elemental impurities) in GDOis 5 ppm. Without being bound by theory, the common presence of thesemetal components and their sequestration in the various aspects of thepresent invention may be at least partially responsible for theadditional stability observed. However, a more common issue may be thepresence of iron ions, which may be absorbed from iron-based alloymaterials used in handling/storage of the materials.

Component b)—Phospholipid

Optional component “b” in the preferred lipid matrices of the presentinvention is at least one phospholipid. It is known from WO2016/066655that lipid slow-release matrices based on triacyl lipids can form depotcompositions on exposure to aqueous fluids without the need for aphospholipid component to be present, though a phospholipid may also bepresent. Thus, in one embodiment component a) comprises, consists orconsists essentially of a triacyl lipid(s) and component b) is optional.However, if component a) is greater than 50% mono-acyl or diacyl lipids,or a tocopherol, or mixtures of any of these components, then aphospholipid component b) will preferably be present. In one embodiment,component a) is less than 50% (e.g. 0 to 45%) triacyl lipid (based onthe total amount of component a)) and component b) is present (e.g. at20 to 80 wt % of the pre-formulation).

When present, preferable ranges of component b) are 20-80 wt. % of thepre-formulation, preferably 30-70 wt. %, more preferably 33-55% (e.g.35-55%), particularly 38 to 43%. When component b) is present, ratios ofa:b are typically 40:60 to 70:30, preferably 45:55 to 55:45 and morepreferably 40:60 to 54:46, such as 45:55 to 54:46 or 47:53 to 53:47.Ratios of around 50:50 (e.g. 49:51 to 51:49) are highly effective incertain embodiments.

Preferred phospholipid polar “head” groups include phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol.Most preferred are phosphatidyl choline (PC) and phosphatidylethanolamine (PE), especially PC. As with component a), this componentcomprises a polar head group and at least one non-polar tail group. Thedifference between components a) and b) lies principally in the polargroup. The non-polar portions may thus suitably be derived from thefatty acids or corresponding alcohols considered above for component a).The phospholipid will contain two non-polar groups. Again, C18 groupsare preferred and may be combined with any other suitable non-polargroup, particularly C16 groups.

The phospholipid portion may be derived from a natural source. In thecase of PC, suitable sources of phospholipids include egg, heart (e.g.bovine), brain, liver (e.g. bovine) and plant sources including soybean.Such sources may provide one or more constituents of component b, whichmay comprise any mixture of phospholipids. Any single PC or mixture ofPCs from these or other sources may be used, but mixtures comprising soyPC or egg PC are highly suitable. The PC component preferably containsat least 50% soy PC or egg PC, more preferably at least 75% soy PC oregg PC and most preferably essentially pure soy PC or egg PC.

In one embodiment applicable to all aspects of the invention, componentb) comprises PC. Preferably the PC is derived from soy. Preferably thePC comprises 18:2 fatty acids as the primary fatty acid component with16:0 and/or 18:1 as the secondary fatty acid components. These arepreferably present in the PC at a ratio of between 1.5:1 and 6:1. PChaving approximately 60-65% 18:2, 10 to 20% 16:0, 5-15% 18:1, with thebalance predominantly other 16 carbon and 18 carbon fatty acids ispreferred and is typical of soy PC.

In an alternative but equally preferred embodiment, the PC component maycomprise synthetic dioleoyl PC (DOPC). The use of DOPC may provideincreased stability and so will be particularly preferable forcompositions needing to be stable to long term storage, and/or having along release period in vivo. In this embodiment the PC componentpreferably contains at least 50% synthetic dioleoyl PC, more preferablyat least 75% synthetic dioleoyl PC and most preferably essentially puresynthetic dioleoyl PC. Any remaining PC is preferably soy or egg PC asabove.

Since the pre-formulations of the invention are to be administered to asubject, possibly with the inclusion of an active agent, it is importantthat the components are biocompatible. In this regard, the preferredlipid matrices for use in the pre-formulations of the present inventionare highly advantageous since tocopherol, PC and acyl glycerols,particularly DAGs, are well tolerated and are broken down in vivo intocomponents that are naturally present in the mammalian body.

It will be appreciated that component b) may include unavoidable traceimpurities of heavy metals. According to the certificates of analysisfor commercially available soy PC (e.g. from Lipoid), a typical maximumconcentration of heavy metals (or elemental impurities) in soy PC is 10ppm.

Synthetic or highly purified PCs, such as dioleoyl phosphatidyl choline(DOPC) are highly appropriate as all or part of component b). Thesynthetic dioleoyl PC is most preferably1,2-dioleoyl-sn-glycero-3-phosphocholine, and other synthetic PCcomponents include DDPC (1,2-Didecanoyl-sn-glycero-3-phosphocholine);DEPC(1,2-Dierucoyl-sn-glycero-3-phosphocholine);DLOPC(1,2-Dilinoleoyl-sn-glycero-3-phosphocholine);DLPC(1,2-Dilauroyl-sn-glycero-3-phosphocholine);DMPC(1,2-Dimyristoyl-sn-glycero-3-phosphocholine);DOPC(1,2-Dioleoyl-sn-glycero-3-phosphocholine);DPPC(1,2-Dipalmitoyl-sn-glycero-3-phosphocholine);DSPC(1,2-Distearoyl-sn-glycero-3-phosphocholine);MPPC(1-Myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine);MSPC(1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine);PMPC(1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine);POPC(1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine);PSPC(1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine);SMPC(1-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine);SOPC(1-Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine); andSPPC(1-Stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine), or anycombination thereof.

A particularly favoured combination of components a) and b) are GDO withPC, especially GDO with soy PC and/or DOPC. Appropriate amounts of eachcomponent suitable for the combination are those amounts indicatedherein for the individual components in any combination. This appliesalso to any combinations of components indicated herein, where contextallows.

Component c)—Biocompatible Organic Solvent

Component c) of the pre-formulations of the invention is at least onebiocompatible organic solvent. Since the pre-formulation is to generatea depot composition following administration (e.g. in vivo), typicallyupon contact with excess aqueous fluid, it is desirable that thissolvent be tolerable to the subject and be capable of mixing with theaqueous fluid, and/or diffusing or dissolving out of the pre-formulationinto the aqueous fluid. Solvents having at least moderate watersolubility are thus preferred. As will be described hereinafter,component c) may include a polar co-solvent.

Component c) comprises or consists of at least one solvent selected fromthe group consisting of: alcohols, amines, amides or esters. Preferablycomponent c) comprises at least a mono-alcoholic solvent. Mostpreferably component c) comprises ethanol, propanol, iso-propanol, ormixtures thereof. It is particularly preferred the component c)comprises or consists of ethanol. Component c) may comprise or consistof a mono-alcoholic solvent, preferably ethanol, and a polar co-solvent.Mixtures comprising or consisting of ethanol and propylene glycol arealso highly preferred.

The amount of component c) in the pre-formulation will have aconsiderable effect upon several features. In particular, the viscosityand the rate (and duration) of release may alter significantly with thesolvent level. The amount of solvent will thus be at least sufficient toprovide a low viscosity mixture but will additionally be determined soas to provide the desired release rate. Typically a level of 1 to 30%,particularly 2 to 20% solvent will provide suitable release andviscosity properties. In some embodiments, levels of 2 to 18%, such as 2to 16%, especially 2 to 15% are preferred.

As indicated above, the amount of component c) in the pre-formulationsof the invention will be at least sufficient to provide a low viscositymixture (e.g. a molecular solution) of components a), c) and ii)(components b) and d) being optional as described herein), and will beeasily determined for any particular combination of components bystandard methods.

The phase behaviour may be analysed by techniques such as visualobservation in combination with polarized light microscopy, X-rayscattering and diffraction techniques, nuclear magnetic resonance, andcryo-transmission electron microscopy (cryo-TEM) to look for solutions,L₂ or L₃ phases, or liquid crystalline phases or as in the case ofcryoTEM, dispersed fragments of such phases. Viscosity may be measureddirectly by standard means. As described above, an appropriate practicalviscosity is that which can effectively be syringed and particularlysterile filtered. This will be assessed easily as indicated herein.

A highly preferred combination for components a), b) and c) is GDO, soyPC and ethanol, especially GDO, soy PC and mixtures of ethanol andpropylene glycol. As indicated above, appropriate amounts of eachcomponent suitable for the combination are those amounts indicatedherein for the individual components, in any combination.

It is preferable that little or none of component c) contains halogensubstituted hydrocarbons since these tend to have lowerbiocompatibility.

Component c) as used herein may be a single solvent or a mixture ofsuitable solvents but will generally be of low viscosity. The viscosityof the “low viscosity” solvent component c) (single solvent or mixture)should typically be no more than 18 mPas at 20° C. This is preferably nomore than 15 mPas, more preferably no more than 10 mPas and mostpreferably no more than 7 mPas at 20° C.

It is described in WO2012/160213 that the addition of a polar solvent inaddition to a mono-alcoholic solvent results in numerous advantagesincluding reduced viscosity and reduced active agent burst profile. Inaddition to the preferred aspects described previously for component c),in one particularly preferred embodiment component c) comprises amono-alcoholic solvent and a polar co-solvent. The term “polarco-solvent” as used herein defines a solvent having a dielectricconstant (diel) of at least 28 at 25° C., more preferably at least 30 at25° C. but is not water or any aqueous fluid. Highly suitable examplesinclude propylene glycol (diel ˜32), and N-methyl-2-pyrrolidone (NMP,diel ˜32). The preferred levels of component c) recited herein applyequally to mixtures of mono-alcoholic solvent and a polar co-solventunless context permits otherwise.

In a particularly preferred embodiment component c) comprises, consistsessentially of, or consists of a mixture of a mono-alcoholic solvent anda polar co-solvent. The polar co-solvent may in one embodiment be adi-alcoholic C3-C6 organic solvent, i.e. a C3-C6 organic solventcomprising two hydroxy groups. The di-alcoholic solvent is preferablypropylene glycol. When present, a polar co-solvent is included at alevel of 2 to 12 wt. % of the pre-formulation, such as 3 to 10 wt. %,especially 4 to 9 wt. %. This level is counted as part of the rangesrecited above for component c). In an embodiment component c) comprises,consists essentially of, or consists of a mixture of ethanol andpropylene glycol (PG).

Where both an organic mono-alcoholic solvent and a polar co-solvent arepresent, e.g. ethanol and PG, the ratio of mono-alcoholic solvent topolar co-solvent solvent is preferably in the range 20:80 to 70:30,preferably 30:70 to 70:30 (w/w), more preferably 40:60 to 60:40.Approximately equal amounts of mono- and di-alcoholic components arehighly appropriate.

In an especially preferred embodiment component c) is present at a levelof 1 to 30% and comprises, consists or consists essentially of a mixtureof ethanol and PG, wherein the ratio of ethanol to PG (w/w) is in therange of 30:70 to 70:30, preferably 40:60 to 60:40. More preferablycomponent c) is present at a range of 5 to 15 wt % or 8 to 18 wt %, mostpreferably 8-18% wt % and is a mixture of ethanol and PG in a ratio of40:60 to 60:40 (w/w).

For the avoidance of doubt, even where a polar co-solvent is present inthe pre-formulations of the present invention, the total water levelwill remain as described in the various embodiments herein (e.g. 0.1 to1.0 wt %).

Component ii)—Alkylammonium Salt

Component ii) is an alkylammonium salt comprising an anion of EDTA(“ethylenediamine tetraacetic acid” or “edetic acid”) or an anion of anEDTA analogue as described below, and at least one alkylammonium cationof Formula (I):NR¹R²R³R^(4a+)  (I)wherein each R¹-R⁴ is independently H, or a linear or branched C1-10group (as described herein), with the proviso that at least one of R¹-R⁴is not H.

Typically, and preferably, n=1. However, for ammonium salts containingmore than one nitrogen atom, such as ethylenediamine (NH₂CH₂CH₂NH₂) itmay be possible for a mixture of +1 and +2 cations to exist (i.e.NH₂CH₂CH₂NH₃ ²⁺ and NH₃CH₂CH₂NH₃ ²⁺). To a certain extent the formationof polycationic species may be prevented by providing an excess of theprecursor amine as described below.

However, the person skilled in the art will appreciate when theformation of mixed cations is a possibility.

Each of R¹ to R⁴ may be the same or different, with the proviso that atleast one of R¹ to R⁴ is not H. Preferably all of the substituent groupsR¹ to R⁴ which are not H are the same. Preferred cations are thereforeNRH₃ ⁺, NR₂H₂ ⁺ and NR₃H⁺ or NR₄+ wherein the “R” groups are the same.Primary, secondary and tertiary ammonium cations are preferred toquaternary cations as the former can be easily prepared by combining theappropriate amine with EDTA as described below.

Each of R¹ to R⁴ is independently H or a linear or branched C1-10 alkyl,alkenyl or alkynyl group, preferably C1-C5. Most preferably each of R¹to R⁴ is a linear or branched C1-5 alkyl group, especially a linearC1-C5 or C1-C3 alkyl group.

Each R¹ to R⁴ may independently be further substituted with one or moreOH or NH₂ (or NH₃+) groups. In an embodiment, for a substituent Rcontaining m carbon atoms, the substituent may contain a maximum of m−1OH and/or NH2 groups per substituent. For instance, if R1 is C8 then R1may contain up to 7 OH groups, especially one OH unit attached to eachcarbon atom other than the carbon atom directly joined to the ammonium Natom. This embodiment is of particular relevance to the case in whichthe alkylammonium cation is derived from an aminopolyol (e.g. meglumine(MeNHCH₂(CHOH)₄CH₂OH)). As an alternative example, if R1 is C3 then R1may contain up to 2 OH groups, such as serinol (NH₂CH(CH₂OH)₂). In anembodiment at least one of R′—R⁴ is a linear C1-C6 group substitutedwith at least one OH or NH₂ group.

In one embodiment any two of the groups R1 to R4 taken together form aC4-C8, preferably C4-C6 ring, which may optionally contain one or moreexocyclic OH or NH2 groups. If any two of the groups R¹ to R⁴ togetherform a ring then a single endocyclic 0 or NH unit may also be present.In particular, it is envisaged that morpholine salts may be used (i.e.if any two of R¹ to R⁴ together form a six-membered C4 ring containingone endocyclic 0 atom). In this embodiment two of the groups R¹ to R⁴along with N together form a morpholine ring, while the remaining groupsR¹ to R⁴ have the definition above.

Particularly preferred alkylammonium cations include those derived fromN-protonation, or in a less preferred embodiment N-alkylation, of anamine selected from:

-   Ethanolamine “ETA” (NH₂(CH₂CH₂OH));-   Diethanolamine “DiETA” (NH(CH₂CH₂OH)₂);-   meglumine (NH(CH₃)CH₂(CHOH)₄CH2OH));-   tris-hydroxymethylamine “TRIS” (N(CH₂OH)₃);-   ethylenediamine (NH₂CH₂CH₂NH₂); or-   serinol (NH₂CH(CH₂OH)₂).

It is preferred that the mass of the alkylammonium cation of Formula (I)is below 500 amu, preferably below 350, especially below 250 amu. Saltsof EDTA containing the ethanolammonium ion (HOCH₂CH₂NH₃ ⁺) areparticularly preferred in the invention. It is most preferred that theEDTA salt is a salt of EDTA with ethanolamine (ETA), preferably EDTAwith ETA only.

In an embodiment the invention relates to EDTA salts comprising an anionof EDTA and at least one alkylammonium cation of Formula (I) aspreviously described, with the proviso that the alkylammonium cation isnot trimethylammonium, tetramethylammonium, triethylammonium ortetraethylammonium.

The alkylammonium cation is thought to aid in increasing the lipidsolubility the EDTA salt relative to a conventional metal (inorganic)EDTA salt such as disodium EDTA. As EDTA contains four carboxylic acidunits the alkylammonium salt may comprise up to four ammonium cationsand a tetraanionic EDTA anion.

As used herein, the term “EDTA” may represent ethylenediaminetetraaceticacid as such. Alternatively, EDTA as indicated herein may include bothethylenediaminetetraacetic acid itself and EDTA analogues. “EDTA” hereinthus includes “EDTA and analogues thereof” whenever context allows.Suitable EDTA analogues are those containing at least one glycinate unit(i.e. the unit —NCH₂COO—) within the molecule, preferably at least 2, atleast 3 or at least 4 glycinate units. Suitable EDTA analogues include:

Iminodiacetic acid (IDA) —(NH(CH₂CO₂H)₂;

Nitrilotriacetic acid (NTA) —N(CH₂CO₂H)₃;

Pentetic acid* —N(CH₂CO₂H)₂CH₂CH₂N(CH₂CO₂H)CH₂CH₂N(CH₂CO₂H)₂;

Egtazic acid —N(CH₂CO₂H)₂CH₂CH₂OCH₂CH₂OCH₂CH₂N(CH₂CO₂H)₂

NOTA —[N(CH₂CO₂H)CH₂CH₂]₃

DOTA —[N(CH₂CO₂H)CH₂CH₂]₄

* Also known as “DTPA”

In an embodiment the EDTA analogue has the structure indicated inFormula (II) below:

wherein n is 1-10, preferably 1-5, especially 1, 2 or 3;wherein X is CH₂, O or NR₄wherein R₁, R₂, R₃ and R₄ are each individually H or CH₂CO₂H, preferablyCH₂CO₂H; or wherein R₁ and R₃ together represent a covalent bond (i.e.the EDTA analogue is cyclic) and R₂ and R₄ are each individually H orCH₂CO₂H, preferably CH₂CO₂H.

Amounts of EDTA and ratios of EDTA to (d) defined herein apply equallyto EDTA and EDTA analogues. In all embodiments it is preferred that EDTAis used as the counterion in component (ii).

Formation of EDTA Salt

The EDTA salt may be pre-formed and dissolved or dispersed in one ormore of the components prior to forming the mixture, e.g.pre-formulation, or may be formed in situ. In situ formation isgenerally preferred for simplicity of operation. A suitable method forpreparing the alkylammonium EDTA salt involves dissolving EDTA (acidform) and the requisite alkylamine (base) in the solvent component (c),or in a solvent which is a precursor to (or sub-component of) thesolvent component (c), and providing mixing until the solids are fullydissolved. In the case of mixtures other than pre-formulations asdefined herein, the EDTA salt may be pre-formed and dissolved ordispersed in component i).

The inventors have established that as a general rule, for a mono-amineat least 3.0, preferably at least 3.5 (e.g. 3.5 to 10) molar equivalentsof amine (which is a precursor to the ammonium salt) are requiredrelative to the amount of EDTA in order to solubilize the salt in thesolvent component (c). As is described in the examples, the minimumratio between the amine and EDTA necessary to solubilize the salt variesdepending on the specific choice of alkylammonium salt. However, anappropriate molar ratio can be achieved by experimentation by simplyobserving at what molar excess of alkylamine the solid EDTA fullydissolves in the solvent. In an embodiment, a greater thanstoichiometric ratio of amine is added than is formally needed to formthe tetraamonium EDTA salt. For instance, as is described in thefollowing examples, efficient solubilisation of EDTA using TRIS mayrequire 5.0 or more equivalents of amine.

For certain di-amines or tri-amines the molar ratio to achieve adequateEDTA salt solubility may not be as high as for a mono-amine. Forpolyamines (diamines, triamines etc), such as NH₂CH₂CH₂NH₂, the requiredmolar ratio may be lower than that for a mono-amine. Suitable levels forpolyamines may be 2.0 or more (e.g. 2.0 to 4.0), or 2.5 or more. Again,suitable levels can be found by optimisation. As a guide, the molarequivalents of amine discussed above may represent the molar ratio ofmono-amine to EDTA or the ratio of amine moieties to EDTA where theamine (or mixture of amines) has more than one amine moiety in themolecule (either individually or on average for a mixture).

There is no upper limit on the number of equivalents of amine which maybe present, although it will be appreciated that typically no more amineshould be included than is necessary to ensure efficient solubilisation.A typical practical limit may be 20 equivalents, preferably 10equivalents.

The inventors have established that in order to form the alkylammoniumEDTA salt, it is necessary to begin with the acid form of EDTA ratherthan the commonly used disodium EDTA (EDTA(Na)). Neither EDTA (edeticacid) nor EDTA(Na) are soluble in suitable/preferred solvents (e.g.EtOH/PG) without an alkylamine (e.g. ETA), even after several months ofmixing. Surprisingly, EDTA(Na) is insoluble in EtOH/PG even in thepresence of ETA.

A typical procedure for producing the salt therefore involves dissolvingthe free tetraacid EDTA (which may be a hydrate) in the solvent (c), orin a solvent which is a precursor to the solvent component (c), whichcomprises at least a mono-alcoholic solvent such as ethanol, and mayalso comprise a polar co-solvent as previously described, preferably ina mixture of ethanol and PG. The requisite number of equivalents ofalkylamine are then added and the mixture is agitated, e.g. byend-over-end rotation or magnetic stirring until the EDTA is dissolved,as can be established by visual observation. 24 h of mixing is usuallyadequate to ensure efficient solubility, e.g. in the case of theformation of an ETA/EDTA salt.

It is also within the scope of the invention to form the salt in asolvent which is a precursor to the solvent component (c). By“precursor” it is meant that the solvent in which the EDTA salt isformed is not identical to the final composition of solvent component(c), but that the content of solvent(s) in the precursor can be adjustedto arrive at the final composition of the solvent (c) in thepre-formulation. As an example, the salt may be formed in a mixture ofEtOH:PG (1:2) and additional ethanol added during or after saltformation in order to reach a final composition of EtOH:PG (1:1) forcomponent (c).

Ratio of Alkylamine to EDTA

The inventors have surprisingly established that above a certain ratioof alkylamine:EDTA the chemical stability of the active agent in thepre-formulation begins to decrease. This may be a result of reactionbetween the excess alkylamine and the active agent, either directly orvia degradation products. Accordingly, it is preferred that the amountof alkylamine chosen is sufficient to fully solubilize all of the EDTAin the solvent component (c) but is not significantly beyond this level.It is preferred that the amount of alkylamine included is no more than 2times the required level to achieve complete solubility, preferably nomore than 1.5 times, preferably no more than 1.2 times. The amount ofalkylamine necessary to fully solubilize ETDA in the solvent component(c) can be established by the methods described previously.

In an embodiment component (ii) comprises an alkylammonium counterionhaving only one amino or alkylamino group and the ratio of EDTA:thetotal of said alkylammonium counterion and any amine free base thereofin the pre-formulation is 1:≥3.0; preferably 1:≥3.5, most preferably inthe range of 1:3.0 to 1:10.

In an embodiment component (ii) comprises an alkylammonium counterionhaving two or more amino and/or alkylamino groups, wherein the ratio ofEDTA:the total of said alkylammonium counterion and any amine free basethereof in the pre-formulation is 1:≥2.0; preferably in the range of1:2.0 to 1:4.0.

In a particularly preferred aspect the EDTA salt is an ETA salt of EDTA.The inventors have established that in this embodiment in order to fullysolubilise EDTA in the solvent component (c) (e.g. a mixture of EtOH/PG50:50) it is necessary to include around at least 3.5 molar equivalentsof ETA relative to the amount of EDTA. Accordingly, the amount of ETA toEDTA is preferably no more than 7:1. The equivalents of ETA to EDTA arepreferably in the range of 3.5 to 7 (mol/mol), preferably 3.5 to 5, mostpreferably 3.5 to 4.5. Most preferably 4 equivalents of ETA are usedrelative to the amount of EDTA (mol/mol).

Amount of EDTA Salt

The level of alkylammonium EDTA salt is chosen to ensure appropriatestability of the components of the lipid vehicle and active agent (ifany) for the storage duration required and under the chosen storageconditions. Factors to be considered when determining appropriateamounts of alkylammonium EDTA salt include: the reactivity of the lipidcomponents and active agent (if any), the loading of active agent (ifany), the molecular mass of the active agent, storage conditions (oxygencontent, humidity, temperature), the duration of oxidative protectionrequired and the concentration of metal ions present in thepre-formulation (which may catalyse decomposition processes).

In order to suppress the catalytic activity of metals, e.g. Fe, thepre-formulation will typically include the EDTA salt at a level suchthat the ratio of EDTA salt to metal (e.g Fe, especially in the form ofFe(II) and Fe(III) ions) is at least around 2:1 (mol/mol), i.e. the EDTAsalt is present in at least a 2 times molar excess. In a typicalprocedure the molar ratio will be based upon the maximum estimated metalion (especially Fe ion) concentration and EDTA provided in a ratio ofaround 2:1 to this maximum estimate. The result in practice will then be2:3 or greater molar ratio of EDTA to metal (e.g. Fe ions).

The inventors have established that there is a preferred level of EDTA,above which no advantage in terms of oxidation resistance of themixture, e.g. pre-formulation is observed, and indeed the stability maybe somewhat reduced. This is influenced by the amount of metal ions(e.g. Fe ions) present in the formulation as is discussed in detail inthe “Experimental” section. However, in general a suitable amount ofEDTA salt in the pre-formulation (calculated in terms of EDTA free acid)will be 0.001-0.02 wt % (10-200 ppm), preferably 0.001-0.015 wt %(10-150 ppm), especially 0.002-0.015 wt % (20-150 ppm). A particularlypreferred level is 0.005-0.015 wt. % (50-150 ppm), most preferably0.008-0.012 wt. % (80-120 ppm). A level of 100 ppm is suitable forprotecting against up to 10 ppm of metal (iron equivalents) which isreasonable for ensuring appropriate drug product robustness.

In certain embodiments, the levels of EDTA (based on the weight of EDTAalone and not including the amine countercations) may range from 0.001to 0.8 wt % (10 to 8000 ppm), 0.002 to 0.5 wt % (20 to 5000 ppm), 0.005to 0.2 wt % (50 to 2000 ppm) or 0.01 to 0.1 wt % (100 to 1000 ppm) ofthe pre-formulation. In certain embodiments the level of EDTA may rangefrom 0.001 to 0.050 wt % (10 to 500 ppm) of the mixture, e.g.pre-formulation, preferably 0.002 to 0.030 wt % (20 to 300 ppm) of themixture.

The level of alkylamine to be added can be established once the optimumratio of alkylamine to EDTA is found, as described in precedingsections.

In an embodiment the ratio of (ii) to (d) is in the range 1:1 to 1:5000(w/w), preferably 1:1 to 1:500 (w/w), preferably in the range of 1:50 to1:300.

Water Content

The inclusion of EDTA salts containing an alkylammonium ion of Formula(I) allows for an antioxidant to be included in the mixture, e.g.pre-formulation, at low levels of water. It is however extremelydifficult to completely eliminate all traces of water (especially fromthe raw materials). Even if essentially water-free formulations could beachieved, pre-formulations will typically be stored in ready-to-useform, e.g. in syringes and possibly under refrigerated conditions.Syringes are often not completely air-tight meaning that the level ofwater in the pre-formulation may increase to an appreciable level overtime, e.g. over months, even if the initial level of water isinsignificant.

The initial absolute level of water in the mixture, e.g.pre-formulation, is between 0 to 1.0 wt. %. Preferably the water contentis less than 1.0 wt. %, preferably less than 0.8 wt %, preferably lessthan 0.5 wt %. Most preferably, the level of water is in the range of0.1 to 0.9 wt. %, especially 0.2 to 0.8 wt. %. These levels refer to theabsolute level of water and not added levels of water. Any unavoidabletrace of water present within components a), b) or c) is included inthis stated level of water. After 3 months of storage, the absolutewater level is preferably no more than 1.5 wt %. Absolute levels ofwater can be measured by methods well known in the art such as KarlFischer titration. In particular, the water content is preferablymeasured according to the procedure in United States Pharmacopoeia (USP40-NF 35, USP <921> Water determination, Method Ia.

Component d)—Active Agent

The pre-formulations of the present invention may contain one or morepeptide or non-peptide active agents. It is emphasised that thesurprising discovery that the oxidation of lipid pre-formulations havinglow levels of water (no more than 1.0%), and optionally any active agentcontained therein can be reduced by the inclusion of particular EDTAsalts herein described, is of very general applicability and thereforethe nature of the bioactive agent is not particularly critical to theworking of the invention. Indeed, since oxidation of the lipidcomponents is reduced by the method of the invention, advantages of thepresent invention may be obtained independently of the nature or evenpresence of any active agent.

It is envisaged that the invention is applicable to lipidpre-formulations containing any bioactive agent of interest. Bioactiveagents may be any compound having a desired biological or physiologicaleffect, such as a peptide, protein, drug, antigen, nutrient, cosmetic,fragrance, flavouring, diagnostic, pharmaceutical, vitamin, or dietaryagent and will be formulated at a level sufficient to provide an in vivoconcentration at a functional level (including local concentrations fortopical compositions). Most preferred active agents are pharmaceuticalagents including drugs, vaccines, and diagnostic agents. An especiallypreferred class of active agents is somatostatins and somatostatinanalogues.

Examples of drugs which may be delivered by the composition of thepresent invention include, but are not limited to, antibacterial agents,immune modulating agents, including immunostimulants andimmunosuppressants, anticancer and/or antiviral drugs such as nucleosideanalogues, paclitaxel and derivatives thereof, anti inflammatorydrugs/agents, such as non-steroidal anti inflammatory drugs andcorticosteroids, cardiovascular drugs including cholesterol lowering andblood-pressure lowing agents, analgesics, anti-emetics includinghistamine H1, NK1 and 5-HT₃ receptor antagonists, corticosteroids andcannabinoids, antipsychotics and antidepressants including serotoninuptake inhibitors, prostaglandins and derivatives, vaccines, and bonemodulators. Diagnostic agents include radionuclide labelled compoundsand contrast agents including X-ray, ultrasound and MRI contrastenhancing agents. Nutrients include vitamins, coenzymes, dietarysupplements etc.

Particularly suitable active agents include those which would normallyhave a short residence time in the body due to rapid breakdown orexcretion and those with poor oral bioavailability. These includepeptide, protein and nucleic acid based active agents, hormones andother naturally occurring agents in their native or modified forms. Byadministering such agents in the form of a depot composition formed fromthe pre-formulation of the present invention, the agents are provided ata sustained level for a length of time which may stretch to days, weeksor even several months in spite of having rapid clearance rates. Thisoffers obvious advantages in terms of stability and patient complianceover dosing multiple times each day for the same period. In onepreferred embodiment, the active agent thus has a biological half life(upon entry into the blood stream) of less than 1 day, preferably lessthan 12 hours and more preferably less than 6 hours. In some cases thismay be as low as 1-3 hours or less. Suitable agents are also those withpoor oral bioavailability relative to that achieved by injection, forwhere the active agent also or alternatively has a bioavailability ofbelow 20%, or preferably below 2%, especially below 0.2%, and mostpreferably below 0.1% in oral formulations.

The amount of bioactive agent to be formulated with the pre-formulationsof the present invention will depend upon the functional dose and theperiod during which the depot composition formed upon administration isto provide sustained release. Typically, the dose formulated for aparticular agent will be around the equivalent of the normal daily dosemultiplied by the number of days the pre-formulation is to providerelease. Evidently this amount will need to be tailored to take intoaccount any adverse effects of a large dose at the beginning oftreatment and so this will generally be the maximum dose used. Theprecise amount suitable in any case will readily be determined bysuitable experimentation.

In an embodiment the pre-formulation of the invention may comprise oneor more peptide active agents. Peptide active agents may comprise 5 to60 natural and/or synthetic amino acids, especially 5 to 50 or 5 to 40amino acids.

Peptide and protein based active agents include human and veterinarydrugs selected from the group consisting of adrenocorticotropic hormone(ACTH) and its fragments, angiotensin and its related peptides,antibodies and their fragments, antigens and their fragments, atrialnatriuretic peptides, bioadhesive peptides, bradykinins and theirrelated peptides, calcitonin peptides including calcitonin and amylinand their related peptides, vasoactive intestinal peptides (VIP)including growth hormone releasing hormone (GHRH), glucagon, andsecretin, opioid peptides including proopiomelanocortin (POMC) peptides,enkephalin pentapeptides, prodynorphin peptides and related peptides,pancreatic polypeptide-related peptides like neuropeptide (NPY), peptideYY (PYY), pancreatic polypeptide (PPY), cell surface receptor proteinfragments, chemotactic peptides, cyclosporins, cytokines, dynorphins andtheir related peptides, endorphins and P-lidotropin fragments,enkephalin and their related proteins, enzyme inhibitors,immunostimulating peptides and polyaminoacids, fibronectin fragments andtheir related peptides, gastrointestinal peptides,gonadotrophin-releasing hormone (GnRH) agonists and antagonist,glucagon-like peptides 1 and 2, growth hormone releasing peptides,immunostimulating peptides, insulins and insulin-like growth factors,interleukins, luthenizing hormone releasing hormones (LHRH) and theirrelated peptides (which are equivalent to GnRH agonists as describedbelow), melanocortin receptor agonists and antagonists, melanocytestimulating hormones and their related peptides, nuclear localizationsignal related peptides, neurotensins and their related peptides,neurotransmitter peptides, opioid peptides, oxytocins, vasopressins andtheir related peptides, parathyroid hormone and its fragments, proteinkinases and their related peptides, somatostatins and their relatedpeptides, substance P and its related peptides, transforming growthfactors (TGF) and their related peptides, tumor necrosis factorfragments, toxins and toxoids and functional peptides such as anticancerpeptides including angiostatins, antihypertension peptides, anti-bloodclotting peptides, and antimicrobial peptides; selected from the groupconsisting of proteins such as immunoglobulins, angiogenins, bonemorphogenic proteins, chemokines, colony stimulating factors (CSF),cytokines, growth factors, interferons (Type I and II), interleukins,leptins, leukaemia inhibitory factors, stem cell factors, transforminggrowth factors and tumor necrosis factors. An interesting class ofbioactive agents suitable for the invention are peptide hormones,including those of the: glycoprotein hormone family (the gonadotropins(LH, FSH, hCG), thyroid stimulating hormone (TSH); proopiomelanocortin(POMC) family, adrenocorticotropic hormone (ACTH); the posteriorpituitary hormones including vasopressin and oxytocin, the growthhormone family including growth hormone (GH), human chorionicsomatomammotropin (hCS), prolactin (PRL), the pancreatic polypeptidefamily including PP, PYY and NPY; melanin-concentrating hormone, (MCH);the orexins; gastrointestinal hormones and peptides including GLP-1 andGIP; ghrelin and obestatin; adipose tissue hormones and cytokinesincluding leptin, adiponectin, and resistin; natriuretic hormones;parathyroid hormone (PTH); the calcitonin family with calcitonin andamylin; the pancreatic hormones including insulin, glucagon andsomatostatin. All synthetic peptides designed to have similar receptoraffinity spectrums as the above mentioned peptides are also verysuitable for the invention.

A further considerable advantage of the depot compositions of thepresent invention is that active agents are released gradually over longperiods without the need for repeated dosing. The compositions are thushighly suitable for situations where patient compliance is difficult,unreliable or where a level dosage is highly important, such asmood-altering actives, those actives with a narrow therapeutic window,and those administered to children or to people whose lifestyle isincompatible with a reliable dosing regime and for “lifestyle” activeswhere the inconvenience of repeated dosing might outweigh the benefit ofthe active. Particular classes of actives for which this aspect offers aparticular advantage include contraceptives, hormones includingcontraceptive hormones, and particularly hormones used in children suchas growth hormone, anti-addictive agents, and drugs used in treatment ofpoorly compliant populations, such as patients suffering fromschizophrenia, Alzheimer, or Parkinson's disease, anti-depressants andanticonvulsants.

Cationic peptides and proteins are particularly suitable for use where aportion of the pre-formulation comprises an anionic amphiphile such as afatty acid or anionic lipid, including phosphatidic acid,phosphatidylglycerol, phosphatidylserine. In this embodiment, preferredpeptides or proteins include octreotide, lanreotide, calcitonin,oxytocin, interferon-beta and -gamma, interleukins 4, 5, 7 and 8 andother peptides or proteins having an isoelectric point above pH 7,especially above pH 8.

In one preferred aspect of the present invention, the composition of theinvention is such that a reversed micellar cubic (I₂) phase, or a mixedphase including I₂ phase is formed upon exposure to aqueous fluids and apolar active agent is included in the composition. Particularly suitablepolar active agents include peptide and protein actives, oligonucleotides, and small water soluble actives, including those listedabove. Of particular interest in this aspect are the peptide octreotideand other somatostatin related peptides, interferons alpha and beta,glucagon-like peptide 1 and glucagon-like peptide 2 receptor agonists,leuprorelin and other GnRH agonists, abarelix and other GnRHantagonists, granisetron and ondansetron and other 5-HT₃ receptorantagonists.

GnRH Analogues

GnRH analogues form one particular class of active agents which may beincluded in formulations of the present invention.

Gonadotropin-releasing hormone agonists (GnRH agonists) are syntheticpeptides modelled after the hypothalamic neurohormone GnRH thatinteracts with the gonadotropin-releasing hormone receptor to elicit itsbiologic response, the release of the pituitary hormones folliclestimulating hormone (FSH) and luteinizing hormone (LH). GnRH agonistsare useful in treatment of cancers that are hormonally sensitive andwhere a hypogonadal state decreases the chances of a recurrence. Thusthey are commonly employed in the medical management of prostate cancerand have been used in patients with breast cancer. Other indicationareas include treatment of delaying puberty in individuals withprecocious puberty, management of female disorders that are dependent onestrogen productions. In addition, women with menorrhagia,endometriosis, adenomyosis, or uterine fibroids may receive GnRHagonists to suppress ovarian activity and induce a hypoestrogenic state.

Gonadotropin-releasing hormone receptor agonists (GnRH-RAs), such asleuprolide (or leuprorelin), goserelin, histrelin, triptorelin,buserelin, deslorelin, nafarelin and related peptides are used orindicated for the treatment of a variety of conditions where they aretypically administered over an extended period. GnRH-RAs form apreferred group of active agents for use in the present invention.

GnRH itself is a post-translationally modified decapeptide of structurepyro-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂ (GnRH-I). Two naturalvarients are also known, GNRH-II having 5-His, 7-Trp, 8-Tyrsubstitutions and GnRH III having 7-Trp, 8-Leu. Several peptideanalogues with agonistic properties are known, most of which have the10-Gly-NH₂ replaced with N-Et-NH₂. Fertirelin has 10-Gly to N-Et-NH₂substitution only, while analogues having additional substitutions overGnRH-I include Leuprorelin (Leuprolide), (6-D-Leu), Buserelin(6-Ser(Bu^(t))), Histrelin (6-d-His(Imbz1)), Deslorelin (6-d-Trp).Another common nona-peptide agonist is Goserelin which is substitutedwith 6-Ser(Bu^(t)) and has 10-Gly-NH₂ replaced by AzaGly-NH₂. Narafelin(6-d-Nal) and Triptorelin (6-d-Trp) both retain the 10-Gly-NH₂ group.The structures of the two most common GnRH agonists (Leuprolide andGoserelin) are shown below as acetate salts.

Leuprolide: pyro-Glu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-N-Et-NH₂(acetate)

Goserelin: pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu^(t))-Leu-Arg-Pro-Azgly-NH₂(acetate)

A small number of GnRH antagonists are also known, again based on theGnRH-I structure. These include Abarelix(D-Ala-D-Phe-D-Ala-Ser-Tyr-D-Asp-Leu-Lys(^(i)Pr)-Pro-D-Ala), Antarelix(D-Nal-D-Phe-D-Pal-Ser-Phe-D-Hcit-Leu-Lys(^(i)Pr)-Pro-D-Ala); Cetrorelix(D-Nal-D-Phe-D-Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala), Ganirelix(D-Nal-D-Phe-D-Pal-Ser-Tyr-D-hArg-Leu-HArg-Pro-D-Ala), Itrelix(D-Nal-D-Phe-D-Pal-Ser-NicLys-D-NicLys-Leu-Lys(^(i)Pr)-Pro-D-Ala) andNal-Glu (D-Nal-D-Phe-D-Pal-Ser-D-Glu-D-Glu-Leu-Arg-Pro-D-Ala).

Administration of single doses of a GnRH agonist, such as leuprolide,stimulates pituitary release of gonadotropins (i.e., LH and FSH),resulting in increased serum LH and FSH concentrations and stimulationof ovarian and testicular steroidogenesis. Transient increases in serumtestosterone and dihydrotestosterone (DHT) in males and in serum estroneand estradiol concentrations in premenopausal females are observedduring initial therapy with single daily doses of the drug.

Although the effect of a potent GnRH agonist during short-term and/orintermittent therapy is stimulation of steroidogenesis, the principaleffect of the drug in animals and humans during long-term administrationis inhibition of gonadotropin secretion and suppression of ovarian andtesticular steroidogenesis. The exact mechanism(s) of action has notbeen fully elucidated, but continuous therapy with a GnRH agonistapparently produces a decrease in the number of pituitary GnRH and/ortesticular LH receptors, resulting in pituitary and/or testiculardesensitization, respectively. The drug does not appear to affectreceptor affinity for gonadotropins. Leuprolide's mechanism of actionmay also involve inhibition and/or induction of enzymes that controlsteroidogenesis. Other mechanisms of action may include secretion of anLH molecule with altered biologic activity or impairment of normalpulsatile patterns of LH and FSH secretion.

A number of serious medical indications are related to and/or affectedby the concentration of gonadal steroid hormones. These include certainneoplastic diseases, including cancers, especially of the breast andprostate, and benign prostatic hypertrophy; premature or delayed pubertyin adolescents; hirsuitism; alzheimer's disease; and certain conditionsrelating to the reproductive system, such as hypogonadism, anovulation,amenorrhea, oligospermia, endometriosis, leiomyomata (uterine fibroids),premenstrual syndrome, and polycystic ovarian disease. Control of thissystem is also important in in vitro fertilisation methods.

Although treatment with a GnRH agonist might be expected to exacerbateconditions affected by gonadal steroid hormone concentration, thedown-regulation effect discussed above results in the decrease of thesehormones to castrate level if therapy is continued for around 2 weeks orlonger. As a result, hormone-receptive tumours such as certain prostateand breast cancer, as well as precocious puberty and many of the otherconditions mentioned above can be improved or palliated by long-termGnRH agonist therapy.

In an embodiment, pre-formulations of the present invention contain oneor more GnRH analogues. Since GnRH is a peptide hormone, typical GnRHanalogues will be peptides, especially of 12 or fewer amino acids.Preferably such peptides will be structurally related to GnRH I, IIand/or III, and/or one or more of the known analogues, including thoselisted here. Peptides may contain only amino acids selected from those20 α-amino acids indicated in the genetic code, or more preferably maycontain their isomers and other natural and non-natural amino acids,(generally α, β or γ amino acids) and their analogues and derivatives.Preferred amino acids include those listed above as constituents of theknown GnRH analogues.

Amino acid derivatives are especially useful at the termini of thepeptides, where the terminal amino or carboxylate group may besubstituted by or with any other functional group such as hydroxy,alkoxy, carboxy, ester, amide, thio, amido, amino, alkyl amino, di- ortri-alkyl amino, alkyl (by which is meant, herein throughout C₁-C₁₂alkyl, preferably C₁-C₆ alkyl e.g. methyl, ethyl, n-propyl, isopropyl,n-butyl, iso-, sec- or t-butyl etc.), aryl (e.g phenyl, benzyl, napthyletc) or other functional groups, preferably with at least one heteroatomand preferably having no more than 10 atoms in total, more preferably nomore than 6.

Particularly preferred GnRH analogues are constrained peptides of 6 to12 alpha-amino acids, of which particular examples include thoseindicated above, and particularly leuprolide and goserelin, of thesequences indicated above.

By “GnRH analogues”, as used herein is indicated any GnRH agonist orantagonist, preferably peptides, peptide derivatives or peptideanalogues. Peptide derived GnRH agonists are most preferred, such asthose indicated above and especially leuprolide or goserelin.

Where present, the GnRH analogue will generally be formulated as 0.02 to12% by weight of the total pre-formulation (based on the amount of freebase). Typical values will be 0.1 to 10%, preferably 0.2 to 8% and morepreferably 0.5 to 6%. A GnRH analogue content of around 1-5% is mostpreferable.

Doses of the GnRH analogue suitable for inclusion in thepre-formulation, and thus the volume of formulation used will dependupon the release rate (as controlled, for example by the solvent typeand amount use) and release duration, as well as the desired therapeuticlevel, the activity of the specific agent, and the rate of clearance ofthe particular active chosen. Typically an amount of 0.1 to 500 mg perdose would be suitable for providing a therapeutic level for between 7and 180 days. This will preferably be 1 to 200 mg. For leuprolide orgoserelin, the level will typically be around 1 to 120 mg (e.g. for a 30to 180 day duration). Preferably, the amount of leuprolide will bearound 0.02 to 1 mg per day between injections, for depots designed forrelease over 30 days to 1 year, preferably 3 to 6 months. Evidently, thestability of the active and linearity of the release rate will mean thatthe loading to duration may not be a linear relationship. A depotadministered every 30 days might have, for example 2 to 30 mg or a 90day depot have 6 to 90 mg of active, such as one of the GnRH analoguesindicated herein.

Somatostatin Analogues

Somatostatin analogues form one particular class of active agents whichmay be included in formulations of the present invention. Somatostatins(Growth Hormone Release Inhibiting Factors, SSTs) are natural peptidehormones with a wide distribution in animals, acting asneurotransmitters in the central nervous system, and having diverseparacrine/autocrine regulatory effects on several tissues. Twobiologically active products are known in higher species, SST-14 andSST-28, the latter being a congener of SST-14 extended at theN-terminus.

SST-14 is a 14 residue cyclic peptide hormone having the sequenceAla-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys, where the twocysteine residues are connected by a disulphide bridge to generate atype II β-turn at the key binding sequence of Phe-Trp-Lys-Thr. Thebiological half-life of natural SST-14 is very short (1-3 minutes) andso it is not, in itself, a viable therapeutic in current formulations,but an increasing number of somatostatin receptor agonists are becomingavailable with higher activities and/or longer clearance times in vivo.

Somatostatin receptor agonists (SRAs), such as SST-14, SST-28,octreotide, lanreotide, vapreotide, pasireotide (SOM 230) and relatedpeptides, are used or indicated in the treatment of a variety ofconditions where they are typically administered over an extendedperiod. SRAs form a preferred group of active agents for use in thepresent invention.

Octreotide, for example, is the synthetic octapeptide with sequenceD-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (2-7 disulphide bridge) and istypically administered as an acetate salt. This SST-14 derivativeretains the key Phe-(D)Trp-Lys-Thr β-turn required for in vivo SST-likeactivity but, in contrast to the natural hormone, has a terminalhalf-life of around 1.7 hours. Octreotide is used in treatment ofconditions including carcinoid tumours and acromegaly, and is typicallyadministered over a sustained period of weeks, or more commonly manymonths or years. Somatostatin receptor agonists are of particularinterest for the treatment of many different types of cancers since awide variety of tumours are found to express somatostatin receptors(SSTRs). There are five known types of SSTRs (SSTR1-SSTR5), showingequally high affinity for SST-14. The most investigated somatostatinreceptor agonists, including octreotide, show high selectivity for SSTR2and SSTR5; thus, octreotide is of particular interest for the treatmentof tumours expressing these types of receptors.

The most common “simple” formulation of Octreotide is “Sandostatin”(RTM) from Novartis. This is an aqueous solution for subcutaneous (s.c)injection, and a 100 μg dose reaches a peak concentration of 5.2 ng/mlat 0.4 hours post injection. The duration of action can be up to 12hours but s.c. dosing is generally carried out every 8 hours. Evidently,s.c. injection 3 times daily for periods of months or years is not anideal dosing regime.

Following a single subcutaneous dose of pasireotide, human plasma levelstypically peak quickly, at around 15 minutes to 1 hour after dosing,with an initial half-life of 2-3 hours following that peak. Althoughclearance half-life is greater for later phases of the decline, it isclear that the Cmax/Cave for such a delivery will be rather high.

Pasireotide LAR is a long acting formulation of pasireotide whichaddresses some of the above issues. However, this is a polymermicroparticle based system with the inherent limitations of such asystem, as are known in the art and described herein above.

Carcinoid tumours are intestinal tumour arising from specialised cellswith paracrine functions (APUD cells). The primary tumour is commonly inthe appendix, where it is clinically benign. Secondary, metastatic,intestinal carcinoid tumours secrete excessive amounts of vasoactivesubstances, including serotonin, bradykinin, histamine, prostaglandins,and polypeptide hormones. The clinical result is carcinoid syndrome (asyndrome of episodic cutaneous flushing, cyanosis, abdominal cramps, anddiarrhea in a patient with valvular heart disease and, less commonly,asthma and arthropathy). These tumours may grow anywhere in thegastrointestinal tract (and in the lungs) with approximately 90% in theappendix. The remainder occurs in the ileum, stomach, colon or rectum.Currently, treatment of carcinoid syndrome starts with i.v. bolusinjection followed by i.v. infusion. When sufficient effect on symptomshas been established, treatment with a depot formulation of octreotideformulated in ploy lactic-co-glycolic acid (PLGA) microspheres isstarted. However, during the first two weeks or more after injection ofthe depot, daily s.c. injections with octreotide are recommended tocompensate for the slow release from the PLGA spheres.

Certain of the pre-formulations of the present invention contain saltsof one or more somatostatin receptor agonists (which are preferredexamples of the peptide actives, which in turn are intended by anyreference to “active agents” herein). Since SST-14 is a peptide hormone,typical somatostatin receptor agonists will be peptides, especially of14 or fewer amino acids. Preferably such peptides will be structurallyconstrained such as by being cyclic and/or having at least oneintra-molecular cross-link. Amide, ester or particularly disulphidecrosslinks are highly suitable. Preferred constrained peptides willexhibit a type-2 β turn. Such a turn is present in the key region ofsomatostatin. Peptides may contain only amino acids selected from those20 α-amino acids indicated in the genetic code, or more preferably maycontain their isomers and other natural and non-natural amino acids,(generally α, β or γ, L- or D-amino acids) and their analogues andderivatives. The term “somatostatin receptor agonist” as used herein mayoptionally also encompass SST-14 and/or SST-28, since these are viablepeptide actives when formulated as salts in the very high performanceslow-release formulations described herein.

Amino acid derivatives and amino acids not normally used for proteinsynthesis are especially useful at the termini of the peptides, wherethe terminal amino or carboxylate group may be substituted by or withany other functional group such as hydroxy, alkoxy, ester, amide, thio,amino, alkyl amino, di- or tri-alkyl amino, alkyl (by which is meant,herein throughout C₁-C₁₈ alkyl, preferably C₁-C₈ alkyl e.g. methyl,ethyl, n-propyl, isopropyl, n-butyl, iso-, sec- or t-butyl etc.), aryl(e.g phenyl, benzyl, napthyl etc) or other functional groups, preferablywith at least one heteroatom and preferably having no more than 10 atomsin total, more preferably no more than 6.

Particularly preferred somatostatin receptor agonists are constrainedpeptides of 6 to 10 α-amino acids, of which particular examples includeoctreotide, lanreotide (of sequenceNH₂-(D)Naph-Cys-Tyr-(D)Trp-Lys-Val-Cys-Thr-CONH₂ and its cyclicderivative of sequence NH₂-(D)Naph-Cys-Tyr-(D)Phe-Lys-Val-Cys-Thr-CONH₂both having a Cys-Cys intramolecular disulphide crosslink), pasireotide(aka SOM 230) and vapreotide. When present, the somatostatin receptoragonist will generally be formulated as 0.1 to 12% by weight of thetotal formulation (based on the amount of free base). Typical valueswill be 0.1 to 10%, 0.5 to 9%, preferably 1 to 8% and more preferably 1to 7%. A somatostatin receptor agonist content of 2-6% is mostpreferable. Doses of the somatostatin receptor agonist suitable forinclusion in the formulation, and thus the volume of formulation used,will depend upon the release rate (as controlled, for example by thesolvent type and amount use) and release duration, as well as thedesired therapeutic level, the activity and the rate of clearance of theparticular active chosen. Typically an amount of 1 to 500 mg per dosewould be suitable for providing a therapeutic level for between 7 and 90days. This will preferably be 5 to 300 mg. For octreotide, the levelwill typically be around 10 to 180 mg (e.g. for a 30 to 90 dayduration). Preferably, the amount of octreotide will be around 0.2 to 3mg per day between injections. Thus a depot administered every 30 dayswould have 6 to 90 mg or a 90 day depot have 18 to 270 mg of octreotide.

For Pasireotide, the dosage would typically be an amount of around 0.05to 40 mg per week of depot duration, preferably 0.1 to 20 mg per weekduration (e.g. 1 to 5 mg per week) for a duration of 1 to 24 weeks,preferably 2 to 16 (e.g. 3, 4, 8, 10 or 12) weeks. In an alternativeembodiment the pre-formulation may be formulated for dosing weekly (e.g.every 7±1 days). A total dose of 0.05 to 250 mg of Pasireotide per dosewould be suitable for providing a therapeutic level for between 7 and168 days. This will preferably be 0.1 to 200 mg, e.g. 0.2 to 150 mg, 0.1to 100 mg, 20 to 160 mg etc. Evidently, the stability of the active andeffects on the release rate will mean that the loading to duration maynot be a linear relationship. A depot administered every 30 days mighthave, for example 0.2 to 20 mg of Pasireotide, or a 90 day depot mighthave 30 to 60 mg of Pasireotide.

Where the salt of a peptide active agent, such as an SRA, is used in theformulations of the present invention, this will be a biologicallytolerable salt. Suitable salts include the acetate, pamoate, chloride orbromide salts. The chloride salt is most preferred.

Other Active Agents

In another embodiment the pre-formulation comprises an active agentwhich is not a somatostatin or a somatostatin analogue. For example, thepeptide active agent may be a peptide which does not interact as eitheragonist or antagonist at any of the SST(1) to SST(5) receptors(especially the corresponding human receptors).

Typically, such pre-formulations will not contain any somatostatin or asomatostatin analogue active agent. That is to say, an active agent ispresent which does not fall within the scope of somatostatin analoguesdescribed in the preceding section. In particular, in this embodimentthe pre-formulation may comprise an active agent which is not selectedfrom endogenous somatostatins, SST-14, SST-28, octreotide, lanreotide,vapreotide or pasireotide or salts thereof. These peptides arepreferably excluded from the pre-formulations of this embodiment. It ispreferred that the pre-formulation is free of somatostatins,somatostatin receptor agonists and somatostatin analogues.

Other active agents which may be contained in pre-formulations of theinvention include:

GnRH antagonists, e.g. cetrorelix, ganirelix, abarelix, degarelix;

GLP-1 and analogues thereof, e.g. GLP-1(7-37), GLP-1(7-36) amide,liraglutide, semaglutide, exenatide, and lixisenatide (AVE0010);

glucagon-like peptide 2 agonists (GLP-2) and analogues thereof, e.g.GLP-2 and elsiglutide (ZP1846);

DPPIV inhibitors; sodium/glucose cotransporter 2 (SGLT2) inhibitors.

Other peptides suitable for the invention include: angiopeptin,angiotensin I, II, III, antileukinate, anti-inflammatory peptide 2,aprotinin, bradykinin, bombesin, calcitonin, calcitriol, cholecystokinin(CCK), colony-stimulating factor, corticotropin-releasing factor,C-Peptide, DDAVP, dermorphin-derived tetrapeptide (TAPS), dynorphin,endorphins, endostatin, endothelin, endothelin-1, enkephalins, epidermalgrowth factor, erythropoietin, fibroblast growth factor, folliclestimulating hormone, follistatin, follitropin, galanin, galanin-likepeptide, galectin-1, gastrin, gastrin-releasing peptide, G-CSF, ghrelin,glial-derived neurotrophic factor, GM-CSF, granulocytecolony-stimulating factor, growth hormone, growth hormone-releasingfactor, hepatocyte growth factor, insulin, insulin-like growth factors-Iand I, interferons, interleukins, leptin, leukemia inhibitory factor,melanocortin 1,2,3,4, melanocyte-stimulating hormone metastin, monocytechemotactic protein-1 (MCP-1), morphiceptin, NEP1-40, neuropeptide Y,neuropeptide W, orexin-A & orexin-B, oxytocin p21-Cipl/WAF-1, TAT fusionprotein, parathyroid hormone, pigment epithelium-derived growth factor(PEDF), peptide, peptide, prorenin handle region, peptide YY (3-36),platelet activating factor, platelet-derived growth factor, prorenindecapeptide, protegrin-1, PR39, prolactin, relaxin, secretin, substanceP, tumor necrosis factor, urocortin, vascular endothelial growth factor,vasoactive intestinal polypeptide, vasopressin.

The short elimination half-life of opioids such as morphine,hydromorphone, and oxycodone require that these agents be administeredfrequently to achieve around-the-clock analgesia, which makes themexcellent candidates for long acting release formulations. Fentanyl andbuprenorphine undergo significant first-pass metabolism and lackssufficient bioavailability after oral administration. Together withtheir high potency, fentanyl and buprenorphine are excellent candidatesfor the long acting injection depot formulation of the invention.Sufentanil, remifentanil, oxymorphone, dimorphone, dihydroetorphine,diacetylmorphine are other potent opioid receptor agonists suitable foruse in the invention.

Buprenorphine is also used for maintenance treatment of opioid addictionas well as potentially also cocaine and amphetamine and met-amphetamineaddiction, where current sublingual buprenorphine formulations sufferfrom low bioavailability, high variability and limited effect duration,resulting in issues with unpredictable dose response and withdrawalsymptoms, particularly in mornings. These issues effectively addressedby using the injection depot formulation of the invention, as areproblems with misuse and misdirection where the need for high sublingualdoses are exploited by injection, where the effect is significantlyhigher for the same dose, thus facilitating misuse of the drug.Similarly, opioid antagonists can be used for treating addiction using aconvenient injection depot system as provided by the invention. Suitableopiate antagonists for use with the invention are naloxone, nalmefene,and naltrexone.

Antipsychotics, including risperidone, iloperidone, paliperidone,olanzapine, asenapine, ziprazidone and aripiprazole are also highlysuitable for the invention in view of the potential for improvedtreatment compliance by patients, as well as by providing stable plasmalevels over time. Similarly, the invention is useful in the treatment ofdementia, Alzheimer's disease and Parkinson's disease, which adverselyaffect cognition. Suitable active ingredients include donepezil,rivastigmine, galantamine, and emantine, rasagilin and pramipexol.

Another group of active agents which may be contained inpre-formulations of the invention are 5HT₃ antagonists. Where the activeagent comprises a 5HT₃ antagonist or second generation 5HT₃ antagonist,this is preferably selected from ondansetron, tropisetron, granisetron,dolasetron, palonosetron, alosetron, cilansetron and/or ramosetron ormixtures thereof. Doses of the 5HT₃ antagonist suitable for inclusion inthe formulation, and thus the volume of formulation used will dependupon the release rate (as controlled, for example by the solvent typeand amount use) and release duration, as well as the desired therapeuticlevel, the activity of the specific agent, and the rate of clearance ofthe particular active chosen. Typically an amount of 1 to 500 mg perdose would be suitable for providing a therapeutic level for between 5and 90 days. This will preferably be 1 to 300 mg. For granisetron, thelevel will typically be around 10 to 180 mg (e.g. for a 3 to 60 dayduration). Preferably, the amount of granisetron will be around 0.2 to 3mg per day between injections, for depots designed for release over 30days to 1 year, preferably 3 to 6 months. Evidently, the stability ofthe active and linearity of the release rate will mean that the loadingto duration may not be a linear relationship. A depot administered every30 days might have, for example 2 to 30 mg or a 90 day depot have 6 to90 mg of active.

In a preferred embodiment the pre-formulation comprises at least oneactive agent which is not a somatostatin receptor agonist. Preferablythe pre-formulation is free from somatostatin receptor agonists. Thus,the pre-formulation may be free of an active agent which interacts aseither agonist or antagonist at any of the SST(1) to SST(5) receptors(particularly in humans).

The term “pre-formulation” herein is a pharmaceutical composition,preferably is a parenteral pharmaceutical composition, more preferablyis an injectable parenteral pharmaceutical composition, even morepreferably is an injectable parenteral pharmaceutical composition forsubcutaneous or intra-muscular application, even more preferably is aninjectable parenteral pharmaceutical composition for subcutaneousapplication.

Optional Additional Components

In one particularly preferred embodiment of the present invention, thecompositions (pre-formulations and resulting depots) do not includefragmentation agents, such as polyethyleneoxide or poly(ethylene glycol)(PEG) fragmentation agent, e.g. a PEG grafted lipid and/or surfactant.

For example, the compositions preferably do not include fragmentationagents such as Polysorbate 80 (P80), or other Polysorbates (e.g.Polysorbate 20), PEGylated phospholipids (PEG-lipids such asDSPE-PEG(2000), DSPE-PEG(5000), DOPE-PEG(2000) and DOPE-PEG(5000)),Solutol HS 15, PEGylated fatty acids (e.g. PEG-oleate), blockco-polymers such as Pluronic® F127 and Pluronic® F68, ethoxylated castoroil derivatives (e.g. Chremophores), PEGylated glyceryl fatty acidesters (such as TMGO-15 from Nikko Chemicals) and PEGylated tocopherols(such as d-alpha tocopheryl poly(ethylene glycol) 1000 succinate knownas Vitamin E TPGS from Eastman.

However, the active agent as a powder (e.g. in the kit of theinvention), as well as active agent dissolved in the lipid formulation,may gain stability (both storage and in vivo stability) by certainstabilising additives. Such additives include sugars (e.g. sucrose,trehalose, lactose etc.), polymers (e.g. polyols such as carboxy methylcellulose), amino acids (such as methionine, glutamate, lysine etc.),lipid-soluble acid components such as HCl, anionic lipids and/or surfaceactive agents (such as dioleoyl phosphatidyl glycerol (DOPG),palmitoyloleoyl phosphatidylglycerol (POPG) and oleic acid (OA)).

Single-dose formats must remain stable and potent in storage prior touse, but are disposable after the single use. It is a remarkable findingthat non-aqueous pre-formulations comprising an alkylammonium EDTA salthave enhanced storage stability at elevated temperatures, such as at 25°C. or even 40° C. This offers advantages in terms of ease oftransportation and storage (no need for refrigeration). It is preferredthat a single dose format has a stability such that after storage for 2months at 25° C. (with air in head space), the assayed active agentconcentration is at least 95% that of the initial assayed active agentconcentration, and after 3 months, the assayed active agentconcentration is at least 90% that of the initial assayed active agentconcentration.

In one preferred embodiment, the pre-formulations, devices and/or kitsof the present invention will be stored at above 10° C. (e.g. at 15 to40° C.), preferably above 20° C., such as at ambient temperature. In acorresponding embodiment, the pre-formulations, devices and/or kits ofthe present invention will not be stored at refrigeration temperatures(e.g. below 10° C. or below 5° C.) such as 1 to 6° C.

It is preferred that a single dose format has a stability such thatafter storage for 2 months at 40° C. (with air in head space), theassayed active agent concentration is at least 85% that of the initialassayed active agent concentration, and after 3 months, the assayedactive agent concentration is at least 80% that of the initial assayedactive agent concentration.

Multi-dose formats must not only remain stable and potent in storageprior to use, but must also remain stable, potent andrelatively/effectively free of bacteria over the multiple-dose useregimen administration period after the first use in which a seal hasbeen compromised. For this reason multi-dose formats often require ananti-microbial or microbial-static agent, e.g. bacteriostatic agent,preservative.

However, the production of preserved pharmaceutical preparationscontaining protein or peptide actives has often proven difficult, aswhen preservatives are used, these give rise to stability problems.Often the proteins are inactivated and aggregates are formed, which maysometimes lead to reported injection site intolerance or immunogenicityto the active. This can be further aggravated by additional excipientsor formulation components.

In one aspect each of the embodiments herein can optionally contain anantimicrobial or microbial-static agent, which includes bacteriostaticagents and preservative. Such agents include benzalkonium chloride,m-cresol, benzyl alcohol or other phenolic preservatives. Typicalconcentrations as known in the art can be used.

Additional components above those mentioned as components i) (includingcomponents a) and c), components b) and d) being optional) and ii) will,where present at all, preferably be present in an amount of 0 to 5%(e.g. 0.01% to 5%) by weight, preferably no more than 2% by weight andmore preferably no more than 1% by weight.

In one embodiment, components a) and b) (allowing for any impurityinherent in the nature of these components) make up at least 95% of thelipid components of the pre-formulations. Preferably at least 99% of thetotal lipid content of the pre-formulation consists of components a) andb). Preferably the lipid component of the pre-formulation consistsessentially of components a) and b).

Administration

The pre-formulations of the present invention are generally formulatedto be administered parenterally. This administration will generally notbe an intra-vascular method but will preferably be subcutaneous (s.c.),intracavitary or intramuscular (i.m.). Typically the administration willbe by injection, which term is used herein to indicate any method inwhich the formulation is passed through the skin, such as by needle,catheter or needle-less (needle-free) injector. It is, however, possibleto take advantage of the high loading and other beneficialcharacteristics of the present formulation in non-parenteralapplications, including topical or systemic application to skin, mucousmembranes, nasal, buccal and/or oral cavities. Preferably, suchnon-parenteral administration is for topical use.

Preferred parenteral administration is by i.m or s.c. injection, mostpreferably by deep s.c. injection. An important feature of thecomposition of the invention is that it can be administered both by i.m.and s.c. and other routes without toxicity or significant local effects.It is also suitable for intracavital administration. The deep s.c.injection has the advantage of being less deep and less painful to thesubject than the (deep) i.m. injection used for some current depots andis technically most suitable in the present case as it combines ease ofinjection with low risk of local side effects. It is a surprisingobservation of the present inventors that the formulations providesustained release of active agent over a predictable time period by bothsubcutaneous and intramuscular injection. This therefore allows the siteof injection to be varied widely and allows the dose to be administeredwithout detailed consideration of the tissue depth at the site ofinjection.

In one embodiment the lipid pre-formulations of the present inventionprovide non-lamellar liquid crystalline depot compositions upon exposureto aqueous fluids, especially in vivo. As used herein, the term“non-lamellar” is used to indicate a normal or reversed liquidcrystalline phase (such as a cubic or hexagonal phase) or the L3 phaseor any combination thereof. The term liquid crystalline indicates allhexagonal, all cubic liquid crystalline phases and/or all mixturesthereof. Hexagonal as used herein indicates “normal” or “reversed”hexagonal (preferably reversed) and “cubic” indicates any cubic liquidcrystalline phase unless specified otherwise. The skilled reader willhave no difficulty in identifying those compositions having appropriatephase behaviour by reference to the description and Examples providedherein, and to WO2005/117830, but the most favoured compositional areafor phase behaviour is where ratio of components a:b are in the regionof 40:60 to 70:30, preferably 45:55 to 55:45 and more preferably 40:60to 54:46. Ratios of around 50:50 (e.g. 49:51 to 51:49) are highlypreferred, most preferably around 50:50.

It is important to appreciate that the pre-formulations of the presentinvention are of low viscosity. As a result, these pre-formulations mustnot be in any bulk liquid crystalline phase since all liquid crystallinephases have a viscosity significantly higher than could be administeredby syringe or similar injecting dispenser. The pre-formulations of thepresent invention will thus be in a non-liquid crystalline state, suchas a solution, L₂ or L₃ phase, particularly solution or L₂. The L₂ phaseas used herein throughout is preferably a “swollen” L₂ phase containinggreater than 5 wt %, preferably greater than 7%, and most preferablygreater than 9% of organic mono-alcoholic solvent (component c) having aviscosity reducing effect.

The pre-formulations described herein are preferably of “low viscosity”.This may be indicated, for example by the ability to be dispensed from a1 ml disposable syringe through a small gauge needle. Preferably, thelow viscosity mixtures can be dispensed through a needle of 19 awg,preferably smaller than 19 gauge, more preferably 23 awg (or mostpreferably even 27 gauge) needle by manual pressure. In a particularlypreferred embodiment, the low viscosity mixture should be a mixturecapable of passing through a standard sterile filtration membrane suchas a 0.22 μm syringe filter. A typical range of suitable viscosities forthe pre-formulations of the invention would be, for example, 1 to 1000mPas, preferably 10 to 800 mPas, more preferably 50 to 750 mPas and mostpreferably 50 to 600 mPas at 20° C.

Upon administration, many of the preferred lipid-based pre-formulationsof the present invention undergo a phase structure transition from a lowviscosity mixture to a high viscosity (generally tissue adherent) depotcomposition. Generally this will be a transition from a molecularmixture, swollen L₂ and/or L₃ phase to one or more (high viscosity)liquid crystalline phases such as normal or reversed hexagonal or cubicliquid crystalline phases or mixtures thereof. Further phase transitionsmay also take place following administration. Obviously, complete phasetransition is not necessary for the functioning of the invention but atleast a surface layer of the administered mixture will form a liquidcrystalline structure. Generally this transition will be rapid for atleast the surface region of the administered formulation (that part indirect contact with air, body surfaces and/or body fluids). This willmost preferably be over a few seconds or minutes (e.g. from 1 second upto 30 minutes, preferably up to 10 minutes, more preferably 5 minutes ofless). The remainder of the composition may change phase to a liquidcrystalline phase more slowly by diffusion and/or as the surface regiondisperses.

The invention is not limited to formulations which undergo a phasechange to a liquid crystalline structure upon administration. A depotcomposition may be formed upon administration by other mechanisms notrequiring the formation of a liquid crystalline phase. For instance, inthe system described in WO2016/066655 the formation of a depotcomposition is not accompanied by a conversion to a liquid crystallinephase.

Without being bound by theory, it is believed that upon exposure toexcess aqueous fluid, the pre-formulations of the invention lose some orall of the organic solvent included therein (e.g. by diffusion) and takein aqueous fluid from the bodily environment (e.g. the in vivoenvironment). For certain lipid pre-formulations as described herein, atleast a part of the formulation preferably generates a non-lamellar,particularly liquid crystalline phase structure. In most cases thesenon-lamellar structures are highly viscous and are not easily dissolvedor dispersed into the in vivo environment. The result is a monolithic“depot” generated in vivo with only a limited area of exposure to bodyfluids. Furthermore, because the non-lamellar structure has large polar,apolar and boundary regions, the lipid depot is highly effective insolubilising and stabilising active agents such as peptides andprotecting these from degradation mechanisms. As the depot compositionformed from the pre-formulation gradually degrades over a period ofdays, weeks or months, the active agent is gradually released and/ordiffuses out from the composition. Since the environment within thedepot composition is relatively protected, the pre-formulations of theinvention are highly suitable for active agents with a relatively lowbiological half-life (see above).

By incorporation of a co-solvent into the pre-formulations, as describedfor the first time in WO2012/160213, it is believed that the rate ofphase transition to a non-lamellar (e.g. liquid crystalline) phase atthe surface of the injected pre-formulation can be enhanced incomparison with compositions containing organic solvents in thesubstantial absence of water. The performance of the resulting depot isthus improved and further control over the release of active agentachieved.

The depot systems formed by the formulations of the present inventionare highly effective in protecting the active agent from degradation andthus allow an extended release period. The formulations of the inventionthus may provide in vivo depots of peptide active agents which requireadministration only once every 5 to 90 days preferably 5 to 60 days,more preferably 6 to 32. Evidently, a longer stable release period isdesirable for patient comfort and compliance, as well as demanding lesstime from health professionals if the composition is not to beself-administered. Where the composition is to be self-administered,patient compliance may be aided by a weekly (e.g. every 7 days,optionally ±1 day), bi-weekly (e.g. every 14 days, optionally ±2 days),or monthly (e.g. every 28 or 30 days (optionally ±7 days) administrationso that the need to administer is not forgotten.

A considerable advantage of the depot precursors of the presentinvention is that they are stable homogeneous phases. That is to say,they may be stored for considerable periods (preferably at least 6months) at room or refrigerator temperature, without phase separation.As well as providing advantageous storage and facile administration,this allows for the dose of active agent (e.g. Somatostatin analogue,e.g. octreotide) to be selected by reference to the species, age, sex,weight, and/or physical condition of the individual subject, by means ofinjecting a selected volume.

The present invention thus provides for methods comprising the selectionof a dosing amount specific to an individual, particularly by subjectweight. The means for this dose selection is the choice ofadministration volume.

In one preferred aspect, the present invention provides apre-formulation comprising a lipid mixture i) comprising components a),b), c), and optionally d), component ii), and 0-1.0% water. The amountsof these components will typically be in the range 20-60% a), 20-60% b),1-30% c) and 0.001-0.8% ii).

The pre-formulations of the present invention are highly advantageous inthat they are stable to prolonged storage in their final “administrationready” form. As a result, they may readily be supplied foradministration either by health professionals or by patients or theircarers, who need not be fully trained health professionals and may nothave the experience or skills to make up complex preparations. This isparticularly important in long-duration, slow-effecting diseases such asdiabetes.

Devices

In a yet further aspect, the present invention provides a disposableadministration device (which is also to include a device component)pre-loaded with a measured dose of a pre-formulation of the presentinvention. Such a device will typically contain a single dose ready foradministration, and will generally be sterile-packed such that thecomposition is stored within the device until administration. Suitabledevices include cartridges, ampoules and particularly syringes andsyringe barrels, either with integral needles or with standard (e.g.luer) fittings adapted to take a suitable disposable needle.

Kits

The pre-filled devices of the invention may also suitably be included inan administration kit, which kit also forms a further aspect of theinvention. In a still further aspect, the invention thus provides a kitfor the administration of at least one active agent, said kit containinga measured dose of a formulation of the invention and optionally anadministration device or component thereof. Preferably the dose will beheld within the device or component, which will be suitable for i.m. orpreferably s.c. administration. The kits may include additionaladministration components such as needles, swabs, etc. and willoptionally and preferably contain instructions for administration. Suchinstructions will typically relate to administration by a route asdescribed herein and/or for the treatment of a disease indicated hereinabove.

The invention provides for a pre-filled administration device asindicated herein and a kit as indicated herein comprising apre-formulation as described herein.

In an alternative aspect of the present invention, the “kit” may containat least two vessels, a first containing a low viscosity mixture of i) alipid mixture comprising components a), c) and optionally b), and ii),as described here, and a second containing a measured dose of at leastone active agent d) as described herein.

Such a “two component kit” may comprise the active agent d) as a powderformulation in one vial or pre-filled syringe and components i) and ii)in a second vial or pre-filled syringe. In the case of two syringes,before injection, the pre-filled syringes are connected and the powdercomprising active agent is mixed with the matrix formulation by movingthe syringe barrels back and forth, forming a solution or suspensionwhich is injected. Alternatively, the liquid lipid formulation is drawnfrom one vial, or is pre-filled into a syringe, and is injected into avial containing powdered active agent (e.g. peptide). This formulationmay subsequently be mixed by hand shaking or other suitablereconstitution method (e.g. vortex mixing etc.). The solvent componentmay be present in either or both vessels (e.g. vials or syringes). Wherethe solvent is at least partially constituted with the active agent,this will generally be in the form of a solution or suspension.

In this aspect, the invention therefore provides a two component kitcomprising

1) a first vessel containing a low viscosity mixture of components a) toc) as described herein;

2) an optional second vessel containing at least one peptide activeagent,

3) an antioxidant component ii) optionally in a third vessel, preferablyin the second vessel, or most preferably in the first vessel;

4) optionally and preferably at least one of:

-   -   5) at least one syringe (which may be one or both of said first        and second vessels);    -   6) a needle for administration, such as those described herein;    -   7) instructions for generation of a composition of the invention        from the contents of the first and second vessels;    -   8) instructions for administration, whereby to form a depot as        described herein.

Preferred Features and Combinations

In combination with the features and preferred features indicatedherein, the mixtures, e.g. pre-formulations, of the invention may haveone or more of the following preferred features independently or incombination:

All proportions indicated herein may optionally be varied by up to 10%of the amount specified, optionally and preferably by up to 5%;

Component a) comprises, consists essentially of or preferably consistsof GDO; Component b) comprises, consists essentially of or preferablyconsists of soy PC; Component c) comprises, consists essentially of orpreferably consists of a 1, 2, 3 or 4 carbon alcohol, preferablyisopropanol or more preferably ethanol;

Component c) includes a polar co-solvent such as propylene glycol;

The pre-formulation does not contain any somatostatin analogue (asdescribed herein);

The pre-formulation has a low viscosity as indicated herein; Thepre-formulation forms a non-lamellar liquid crystalline phase asindicated herein upon in vivo administration;

The pre-formulation generates a depot following in vivo administration,which depot releases at least one active agent at a therapeutic levelover a period of at least 7 days, preferably at least 21 days, morepreferably at least 28 days;

In combination with the features and preferred features indicatedherein, the method(s) of treatment of the present invention may have oneor more of the following preferred features independently or incombination;

The method comprises the administration of at least one formulation withone or more preferred features as indicated above;

The method comprises the administration of at least one formulation asindicated herein by i.m., s.c. (e.g. deep s.c.) injection;

The method comprises administration by means of a pre-filledadministration device as indicated herein;

The method comprises administration through a needle no larger than 20gauge, preferably smaller than 20 gauge, and most preferably 22 gauge,23 gauge or smaller;

The method comprises a single administration every 5 to 90 days,preferably 6 to 32 days (for example 7 days or 28-31 days);

In combination with the features and preferred features indicatedherein, the use(s) of the pre-formulations indicated herein in themanufacture of medicaments may have one or more of the followingpreferred features independently or in combination;

The use comprises the use of at least one formulation with one or morepreferred features as indicated above;

The use comprises the manufacture of a medicament for administration ofat least one formulation as indicated herein by i.m. or s.c. injection;

The use comprises the manufacture of a medicament for administration bymeans of a pre-filled administration device as indicated herein;

The use comprises the manufacture of a medicament for administrationthrough a needle no larger than 20 gauge, preferably smaller than 20gauge, and most preferably 22 gauge, 23 gauge or smaller;

The use comprises the manufacture of a medicament for administrationonce every 5 to 90 days, preferably 5 to 60 days, more preferably 6 to32 days;

In combination with the features and preferred features indicatedherein, the pre-filled devices of the invention may have one or more ofthe following preferred features independently or in combination:

They contain a preferred formulation as indicated herein;

They comprise a needle smaller than 20 gauge, preferably no larger than22 gauge or no larger than 23 gauge;

They contain a homogeneous mixture of a composition of the invention inready-to-inject form.

They contain a formulation of components i) (preferably comprising a),b) and c)) and ii) for combination with an active agent.

They contain a total volume for administration of no more than 5 ml,preferably no more than 3 ml more preferably no more than 1.5 ml.

In combination with the features and preferred features indicatedherein, the kits of the invention may have one or more of the followingpreferred features independently or in combination:

They contain a preferred formulation as indicated herein; They contain apre-filled device as indicated herein; They contain a needle smallerthan 20 gauge, preferably no larger than 22 gauge or no larger than 23gauge;

They contain peptide active agent;

They contain a total volume for administration of no more than 5 ml,preferably no more than 3 ml more preferably no more than 1.5 ml;

They contain instructions for administration by a route and/or at afrequency as indicated herein;

They contain instructions for administration for use in a method oftreatment as described herein;

The Invention will now be further illustrated by reference to thefollowing non-limiting Examples and the attached Figures.

EXAMPLES

Materials

All materials used in the Examples were obtained from commercial sourcesand were of pharmacopoeial grade where applicable or of the highestpurity grade available. The following abbreviations are used throughoutthe Examples:

API Active pharmaceutical ingredient

DiETA Diethanolamine

DTPA Diethylenetriaminepentaacetic (pentetic) acid

EtOH Ethanol (99.7% Ph. Eur)

EDTA Ethylenediaminetetraacetic (edetic) acid (USP/NF)

EDTA(Na) Ethylenediaminetetraacetic acid disodium dihydrate

ETA Ethanolamine (USP/NF)

FeCl₃×6H₂O Iron(III) chloride hexahydrate

GDO Glycerol dioleate (Cithrol GDO HP-SO-(LK) from Croda)

GMO Glycerol monooleate

GOS(Ac) Goserelin acetate

GOS(C1) Goserelin chloride

GRN(0) Granisetron free base

OCT(C1) Octreotide hydrochloride

OXY(Ac) Oxytocin acetate

OXY(C1) Oxytocin chloride

PG Propylene glycol (Ph. Eur)

SbOil Soybean oil

SOM(Ac) Somatostatin-14 acetate

SOM(C1) Somatostatin-14 chloride

SPC Soy phosphatidylcho line (Lipoid S100 from Lipoid)

TRIS Tris(hydroxymethyl)aminomethane

General Procedures

Preparation of EDTA and EDTA(Na) Solutions in EtOH/PG

Samples were prepared by weighing the appropriate amounts of EDTA orEDTA(Na) and alkylamine into glass vials, e.g. 15R vials, followed byaddition of organic solvent or solvent mixture (e.g EtOH/PG (50/50w/w)). Vials were sealed and placed on either a roller mixer byend-over-end rotation at ambient RT or magnetic stirrer. Duringdissolution, vials were visually inspected for undissolved EDTAparticles using ambient and cross-polarized light.

Preparation of FeCl₃×6H₂O Solutions

Samples were prepared by weighing the appropriate amount of FeCl₃×6H₂Ointo sterilized glass vials followed by addition of organic solvent orsolvent mixture. Vials were sealed and placed on a roller mixer byend-over-end rotation at ambient RT until FeCl₃×6H₂O was completelydissolved.

Preparation of SOM(C1)

For the ion-exchange process, approximately 120 g of Dowex 1×2 chlorideform (50-100 mesh) resin was mixed with an equal amount of Milliporewater, added to a 200 mL glass ion-exchange column and left toequilibrate overnight. Next day, prior to ion-exchange the Dowex matrixwas slowly washed with 900 ml Millipore water and the ion-exchangeprocess was initiated. 3.743 g of SOM(Ac) was dissolved in 112.4 gMillipore water. Freshly prepared (within approx. 30 min) SOM(Ac)solution was loaded onto the top of the ion-exchange column. The flow(at approx. 15 s/mL) was initiated and eluate fractions of 50-250 mLeach were collected by continuously rinsing the column with Milliporewater. The eluate fractions with conductivity greater than 50 μS/cm werepooled, transferred into three 1000 mL round-bottom flasks, shell-frozenin EtOH/dry-ice using Rotavapor R-200, placed to cool at −80° C. forabout 1 h and lyophilized overnight for about 36 h. The obtained amountand yield of SOM(C1) were 3.096 g and 82.7%, respectively. The completeexchange of acetate to chloride was confirmed by determination of thetwo anions by indirect HPLC-UV.

Preparation of GOS(C1)

For the ion-exchange process, 34.7995 g of Dowex 1×2 chloride form(50-100 mesh) resin was mixed with 36.6327 g Millipore water, added to a100 mL glass ion-exchange column and left to equilibrate overnight. Nextday, prior to ion-exchange, the Dowex matrix was slowly washed with 700ml Millipore water and the ion-exchange process was initiated. 0.8353 gof GOS(Ac) was dissolved in 13.9534 g Millipore water. Freshly prepared(within approx. 30 min) GOS(Ac) solution was loaded onto the top of theion-exchange column. The flow (at approx. 15 s/mL) was initiated andeluate fractions of 15-50 mL each were collected by continuously rinsingthe column with Millipore water. The eluate fractions with conductivitygreater than 35 μS/cm were pooled, transferred into a 500 mLround-bottom flask, shell-frozen in EtOH/dry-ice using Rotavapor R-200,placed to cool at −80° C. for about 1 h and lyophilized for about 23 h.The obtained amount and yield of GOS(C1) were 0.739 g and 88.5%,respectively. The complete exchange of acetate to chloride was confirmedby determination of the two anions by indirect HPLC-UV.

Preparation of OXY(C1)

For the ion-exchange process, 31.5493 g of Dowex 1×2 chloride form(50-100 mesh) resin was mixed with 42.1860 g Millipore water, added to a100 mL glass ion-exchange column and left to equilibrate overnight. Nextday, prior to ion-exchange, the Dowex matrix was slowly washed with 600ml Millipore water and the ion-exchange process was initiated. 0.7933 gof OXY(Ac) was dissolved in 12.5166 g Millipore water. Freshly prepared(within approx. 30 min) OXY(Ac) solution was loaded onto the top of theion-exchange column. The flow (at approx. 15 s/mL) was initiated andeluate fractions of 15-50 mL each were collected by continuously rinsingthe column with Millipore water. The eluate fractions with conductivitygreater than 25 μS/cm were pooled, transferred into a 500 mLround-bottom flask, shell-frozen in EtOH/dry-ice using Rotavapor R-200,placed to cool at −80° C. for about 1 h and lyophilized overnight forabout 25 h. The obtained amount and yield of OXY(C1) was 0.686 g and86.5%, respectively. The complete exchange of acetate to chloride wasconfirmed by determination of the two anions by indirect HPLC-UV.

Preparation of Lipid Formulations

Lipid placebo formulations were prepared by weighing appropriate amountsof SPC, GDO, EDTA/alkylamine solution, and FeCl₃×6H₂O (when needed)solution into sterilized glass vials. The sealed vials were then placedon a roller mixer at room temperature until mixed completely into clearhomogeneous liquid solution (<24 hours).

API-containing formulations were prepared by adding appropriate amountsof API powder to the lipid placebo formulations in sterilized glassvials. The vials were sealed and placed on a roller mixer at roomtemperature until mixed completely into clear homogeneous liquidsolution (ca. 24 hours).

As an example, EDTA and ETA (at EDTA:ETA molar ratio 1:4) were dissolvedin EtOH/PG (50/50 w/w) mixture. Then, appropriate amounts of SPC, GDO(at SPC/GDO weight ratio 50/50) and EtOH/PG/EDTA/ETA mixture wereweighed into a sterilized 20R glass vial. The sealed vial was thenplaced on a roller mixer at room temperature until mixed completely intoclear homogeneous liquid solution (<24 hours). OCT(C1) powder was thenadded to the lipid formulations in sterilized 15R glass vial at 2.34 wt% concentration. The vial was sealed and placed on a roller mixer atroom temperature until mixed completely into clear homogeneous liquidsolution (24 hours).

Evaluation of Octreotide Stability in Lipid Formulations (TypicalMethod)

Prepared lipid peptide (e.g. octreotide) formulations as above weredivided into sterilized 2R glass vials (0.5 g of formulation per vial).The head space of the vials was ambient air, i.e., no inert atmospheresuch as nitrogen was introduced in the head space. Vials were sealed andplaced in controlled environment storage cabinets at 25° C./60% RH and40° C./75% RH. At predefined sampling points (up to three months ofstorage) two vials of each formulation and storage cabinet werewithdrawn, equilibrated to room temperature for 1 hour and analyzed forpeptide content (assay) using gradient HPLC with UV detection.

It should be noted that the filling procedure and storage conditionsensured forced degradation conditions as the head space was composed ofair rather than inert atmosphere such as nitrogen.

HPLC-UV Determination of Peptides in Lipid Formulations

Determination of peptide (e.g. octreotide, such as octreotide chloride)in lipid formulations was carried out by gradient HPLC with UVdetection. The HILIC analytical column used was a HALO Penta-HILIC 2.7μm, 150×3.0 mm. Quantification was carried out by interpolating thepeptide (e.g. octreotide) peak area obtained in lipid formulationsamples (prepared by dissolving the lipid formulation in a samplesolvent at the required target peptide concentration) into thecalibration curves generated from standard solutions containing knownconcentrations of the corresponding peptide.

A typical mobile phases used (for example with octreotide) consisted ofwater: 2M sodium chloride:acetonitrile:trifluoroacetic acid 384:16:400:1(v/v) (mobile phase A) and water:methanol:acetonitrile:trifluoroaceticacid 20:30:950:1 (v/v) (mobile phase B). The detection was carried outat 220 nm. The sample solvent used was acetonitrile:methanol (1:1, v/v);octreotide eluted after approximately 25.2 min.

Data Presentation

In the example section, in addition to absolute API assay values,results are also in some cases expressed as a Stability Index for APIassay. The Stability Index is calculated as the API assay value in theparticular formulation divided by the API assay value in the referenceformulation. Expressed in this way, Stability Index values greater than1 means improved API stability when compared to the referenceformulation.

Measurement of Vial Headspace Oxygen Concentration

Oxygen concentration in the vial headspace was measured using aPC-controlled PreSens Microx TX3 micro fiber optic oxygen transmitterequipped with a needle-type optical oxygen microsensor (NTH, 140 μm flatbroken tip). Measurements were performed by penetrating the oxygenmicrosensor through the vial rubber stopper into the vial headspace andmeasuring the oxygen concentration until a stable readout was obtained(about 1 min).

Example 1. EDTA Solubility in the Presence and Absence of Alkylamine

0.08 wt % EDTA and EDTA(Na) solutions in EtOH/PG (50/50 w/w) wereprepared in the presence and absence of ETA (Table 1). Dissolution ofEDTA and EDTA(Na) during end-over-end rotation at ambient RT wasassessed by visual inspection (ambient and crossed-polarized light) over27 days. The results show that neither the disodium salt (EDTA(Na)) northe acid form of EDTA is soluble in EtOH/PG without using ETA even after27 days of mixing. The obtained results also show that EDTA(Na) is notsoluble in EtOH/PG even in the presence of ETA whereas the acid form ofEDTA is solubilized in EtOH/PG in the presence of 4 mol ETA per 1 mol ofEDTA already after 24 hours mixing.

TABLE 1 Solubility of 0.08 wt % EDTA and EDTA(Na) in EtOH/PG in thepresence and absence of ETA. ETA/EDTA Observations after mixing forSample No EDTA type (mol/mol) 24 h 27 days Sample 1 Disodium 0.00 Notsoluble Not soluble dihydrate Sample 2 Acid form 0.00 Not soluble Notsoluble Sample 3 Disodium 3.94 Not soluble Not soluble dihydrate Sample4 Acid form 3.95 Soluble Soluble

Example 2. EDTA Solubility as a Function of ETA/EDTA Molar Ratio

Table 2 summarizes results on EDTA solubility at a concentration of 0.38wt % in EtOH/PG solvent mixtures (1/1 wt/wt) as a function of ETA/EDTAmolar ratio. The only sample where EDTA was not fully dissolved was forthe lowest ETA/EDTA molar ratio. In all other samples EDTA was solubleafter 24 h end-over-end rotation mixing at ambient RT. The obtainedresults show that about 3.5 mol of ETA per 1 mol of EDTA is close to therequired minimum amount needed to solubilize EDTA in the non-aqueoussolvent used.

TABLE 2 Solubility of 0.38 wt % EDTA in EtOH/PG as a function ofETA/EDTA molar ratio. ETA/EDTA EDTA solubility Sample ID (mol/mol) (ca24 h mixing) Sample 5 2.83 Not soluble Sample 6 3.49 Soluble Sample 73.90 Soluble Sample 8 3.90 Soluble Sample 9 4.02 Soluble Sample 10 3.99Soluble Sample 11 4.30 Soluble Sample 12 4.24 Soluble Sample 13 4.45Soluble Sample 14 4.48 Soluble Sample 15 4.62 Soluble Sample 16 4.65Soluble

Example 3. EDTA Solubility as a Function of DiETA/EDTA Molar Ratio

Table 3 summarizes EDTA solubility results in EtOH/PG (1/1 wt/wt)solvent mixture at 0.38 wt % EDTA as a function of DiETA/EDTA molarratio after 24 h end-over-end rotation mixing at ambient RT. Theobtained results show that about 4.5 mol of DiETA per 1 mol of EDTA isclose to the required minimum amount needed to solubilize EDTA innon-aqueous solvent used.

TABLE 3 Solubility of 0.38 wt % EDTA in EtOH/PG as a function ofDiETA/EDTA molar ratio. DiETA/EDTA EDTA solubility Sample ID (mol/mol)(ca 24 h mixing) Sample 17 2.14 Not soluble Sample 18 2.68 Not solubleSample 19 3.20 Not soluble Sample 20 3.52 Almost fully soluble Sample 213.97 Soluble or almost fully soluble Sample 22 4.51 Soluble Sample 235.09 Soluble

Example 4. EDTA Solubility as a Function of Ethylenediamine/EDTA MolarRatio

Table 4 summarizes EDTA solubility results in EtOH/PG (1/1 wt/wt)solvent mixture at 0.38 wt % EDTA as a function of ethylenediamine/EDTAmolar ratio after 24 h end-over-end rotation mixing at ambient RT. Theobtained results showed that about 2.5 mol of ethylenediamine per 1 molof EDTA is close to the required minimum amount needed to solubilizeEDTA in non-aqueous solvent used.

TABLE 4 Solubility of 0.38 wt % EDTA in EtOH/PG as a function ofethylenediamine/EDTA molar ratio. Ethylenediamine/EDTA EDTA solubilitySample ID (mol/mol) (ca 24 h mixing) Sample 24 1.96 Not soluble Sample25 2.45 Soluble Sample 26 3.09 Soluble Sample 27 3.46 Soluble Sample 283.92 Soluble Sample 29 4.47 Soluble Sample 30 5.00 Soluble

Example 5. EDTA Solubility as a Function of Serinol/EDTA Molar Ratio

Table 5 summarizes EDTA solubility results in EtOH/PG (1/1 wt/wt)solvent mixture at 0.38 wt % EDTA as a function of serinol/EDTA molarratio after 24 h end-over-end rotation mixing at ambient RT. Theobtained results showed that about 4 mol of serinol per 1 mol of EDTA isclose to the required minimum amount needed to solubilize EDTA innon-aqueous solvent used.

TABLE 5 Solubility of 0.38 wt % EDTA in EtOH/PG as a function ofserinol/EDTA molar ratio. Serinol/EDTA EDTA solubility Sample ID(mol/mol) (ca 24 h mixing) Sample 31 1.88 Not soluble Sample 32 2.36 Notsoluble Sample 33 3.32 Not soluble Sample 34 3.48 Almost soluble Sample35 4.11 Soluble Sample 36 4.77 Soluble Sample 37 5.09 Soluble Sample 385.45 Soluble

Example 6. EDTa Solubility as a Function of TRIS/EDTA Molar Ratio

Table 6 summarizes EDTA solubility results in EtOH/PG (1/1 wt/wt)solvent mixture at 0.38 wt % EDTA as a function of TRIS/EDTA molar ratioafter 7 days end-over-end rotation mixing at ambient RT. The obtainedresults showed that about 5 mol of TRIS per 1 mol of EDTA is close tothe required minimum amount needed to solubilize EDTA in non-aqueoussolvent used.

TABLE 6 Solubility of 0.38 wt % EDTA in EtOH/PG as a function ofTRIS/EDTA molar ratio. TRIS/EDTA EDTA solubility Sample ID (mol/mol) (ca7 days mixing) Sample 39 2.03 Not soluble Sample 40 2.57 Not solubleSample 41 2.96 Not soluble Sample 42 3.52 Not soluble Sample 43 3.97Almost soluble Sample 44 4.49 Almost soluble Sample 45 5.04 SolubleSample 46 4.98 Soluble Sample 47 5.55 Soluble Sample 48 5.98 SolubleSample 49 6.48 Soluble Sample 50 6.97 Soluble Sample 51 7.47 SolubleSample 52 8.03 Soluble

Example 7. Stability of OCT(C1) in Lipid Formulations in the Presence ofEDTA

Lipid formulations containing 2.34 wt % of OCT(C1) in the presence andabsence of 100 ppm of EDTA were prepared according to the compositionsgiven in Table 7. Formulations were divided into sterilized 2R glassvials (0.5 g of formulation per vial), sealed and placed in controlledenvironment storage cabinets at either 40° C./75% RH or 25° C./60% RH.The headspace of the vials was ambient air to ensure forced degradationconditions, i.e., no inert atmosphere such as nitrogen was introduced.At predefined sampling points (up to three months of storage), two vialsof each formulation and storage condition were withdrawn from thecontrolled environment cabinets, equilibrated to room temperature for 1hour and analyzed for peptide content (assay) using gradient HPLC withUV detection.

TABLE 7 OCT(Cl) containing FluidCrystal ® formulation compositions (inwt %) with and without EDTA. Sample ID OCT(Cl) SPC GDO EtOH PG ETA EDTASample 53 2.34 42.33 42.33 6.50 6.50 — — Sample 54 2.34 42.32 42.32 6.506.50 0.01 0.01

Samples of the two formulations were placed on stability as describedunder General Procedures. It should be noted that the filling procedureand storage conditions ensured forced degradation conditions as the headspace was composed of air rather than inert atmosphere such as nitrogen.FIG. 1 presents the octreotide assay at different storage time pointsand storage conditions. As shown in FIG. 1, the presence of 0.01 wt %(100 ppm) of EDTA solubilized in the lipid formulation by the use of0.01 wt % (100 ppm) ETA dramatically enhanced the peptide stability atboth storage conditions.

Example 8. Effect of EDTA Concentration on Peptide Stability

Lipid formulations containing 2.27 wt % of OCT(C1) and differentconcentrations of EDTA were prepared according to the compositions givenin Table 8. Formulations were divided into sterilized 2R glass vials(0.5 g of formulation per vial), sealed and placed in controlledenvironment storage cabinets at either 40° C./75% RH or 25° C./60% RH.The head space of the vials was ambient air to ensure forced degradationconditions, i.e., no inert atmosphere such as nitrogen was introduced.At predefined sampling points (up to six months of storage) two vials ofeach formulation and storage condition were withdrawn from thecontrolled environment cabinets, equilibrated to room temperature for 1hour and analyzed for peptide content (assay) using gradient HPLC withUV detection.

TABLE 8 Formulation compositions with different concentrations of EDTA(all components in wt %) comprising 2.27 wt % OCT(Cl). Sample ID OCT(Cl)SPC GDO EtOH PG ETA EDTA Sample 55 2.27 42.37 42.37 6.50 6.50 — — Sample56 2.27 42.36 42.36 6.50 6.50 0.004 0.005 Sample 57 2.27 42.36 42.366.50 6.50 0.008 0.010 Sample 58 2.27 42.34 42.34 6.50 6.50 0.021 0.025Sample 59 2.27 42.32 42.32 6.50 6.50 0.042 0.050 Sample 60 2.27 42.3042.30 6.50 6.50 0.063 0.075

Samples of the six formulations were placed on stability as describedunder General Procedures. It should be noted that the filling procedureand storage conditions ensured forced degradation conditions as the headspace was composed of air rather than inert atmosphere such as nitrogen.The results are shown in FIG. 2. As shown, the presence of EDTAsolubilized in the lipid formulation with the help of ETA dramaticallyenhanced the peptide stability vs. the reference formulation notcontaining EDTA/ETA. The maximum stabilization effect was achievedwithin the concentration interval 50-250 ppm (0.005-0.025 wt %) EDTA.

Example 9. Long-Term Stability of OCT(C1) in Lipid Formulations in thePresence of EDTA

Lipid formulations containing OCT(C1) in the absence and presence of 100ppm EDTA were prepared according to the compositions given in Table 9.Formulations were divided into sterilized 1 mL 22Gx1/2″ glass syringes(Schott AG) (0.5 g of formulation per syringe), sealed with plunger andplaced in a controlled environment storage cabinet at 25° C./60% RH. Atpredefined sampling points (up to twelve months of storage), twosyringes of each formulation were withdrawn from the controlledenvironment cabinet, equilibrated to room temperature for 1 hour andanalyzed for peptide content (assay) using gradient HPLC with UVdetection.

TABLE 9 OCT(Cl) containing lipid formulation compositions (in wt %)without and with EDTA. The octreotide content corresponds to 20 mg/mLoctreotide free base when corrected for peptide content, purity andformulation density. Sample ID OCT(Cl) SPC GDO EtOH PG ETA EDTA Sample61 2.27 42.37 42.37 6.50 6.50 — — Sample 62 2.27 42.36 42.36 6.50 6.500.008 0.010

FIG. 3 presents the octreotide assay at different storage time points.As shown, the presence of 0.01 wt % (100 ppm) of EDTA solubilized in thelipid formulation with the help of ETA significantly enhanced thelong-term peptide stability in pre-filled syringes at the long-term 25°C./60% RH storage condition.

Example 10. Stability of OCT(C1) in Lipid Formulations in the Presenceof Iron and EDTA

Lipid formulations containing OCT(C1) and different amounts of Fe³⁺ andEDTA were prepared according to the compositions given in Table 10.Formulations were divided into sterilized 2R glass vials (0.5 g offormulation per vial), sealed and placed in a controlled environmentstorage cabinet at 40° C./75% RH. The head space of the vials wasambient air to ensure forced degradation conditions, i.e., no inertatmosphere such as nitrogen was introduced. At 1-month sampling pointtwo vials of each formulation and storage condition were withdrawn fromthe controlled environment cabinet, equilibrated to room temperature for1 hour and analyzed for peptide content (assay) using gradient HPLC withUV detection.

TABLE 10 OCT(Cl) containing FluidCrystal ® formulation compositions (inwt %) with different concentrations of Fe³⁺ and EDTA. The octreotidecontent corresponds to 20 mg/mL octreotide free base when corrected forpeptide content, purity and formulation density. The SPC/GDO weightratio is 50/50 in all formulations. Sample ID OCT(Cl) SPC + GDO EtOH PGEDTA ETA FeCl₃ × 6H₂O* Sample 63 2.27 84.73000 6.50 6.50 — — — Sample 642.27 84.72903 6.50 6.50 — — 0.00097 Sample 65 2.27 84.72758 6.50 6.50 —— 0.00242 Sample 66 2.27 84.72516 6.50 6.50 — — 0.00484 Sample 67 2.2784.72541 6.50 6.50 0.00250 0.00209 — Sample 68 2.27 84.72444 6.50 6.500.00250 0.00209 0.00097 Sample 69 2.27 84.72299 6.50 6.50 0.002500.00209 0.00242 Sample 70 2.27 84.72057 6.50 6.50 0.00250 0.002090.00484 Sample 71 2.27 84.71164 6.50 6.50 0.01000 0.00836 — Sample 722.27 84.71067 6.50 6.50 0.01000 0.00836 0.00097 Sample 73 2.27 84.709226.50 6.50 0.01000 0.00836 0.00242 Sample 74 2.27 84.70680 6.50 6.500.01000 0.00836 0.00484 Sample 75 2.27 84.68409 6.50 6.50 0.025000.02091 — Sample 76 2.27 84.68312 6.50 6.50 0.02500 0.02091 0.00097Sample 77 2.27 84.68167 6.50 6.50 0.02500 0.02091 0.00242 Sample 78 2.2784.67925 6.50 6.50 0.02500 0.02091 0.00484 *0.00097, 0.00242, and0.00484 wt % of FeCl₃ × 6H₂O corresponds to 2, 5, and 10 ppm of Fe³⁺,respectively.

FIG. 4 presents the octreotide assay at 1-month time point as a functionof Fe³⁺ concentration in the presence of different amounts of EDTA. Asevident, with increasing the Fe³⁺ concentration, more EDTA is needed toprotect OCT from degradation. The protection against OCT degradation inthe presence of Fe³⁺ is enhanced with increasing EDTA concentration upto 100 ppm, followed by some decline between 100 and 250 ppm. There isalso a clear correlation between Fe³⁺ concentration and amount of EDTAneeded to suppress the catalytic activity of iron. As shown in FIG. 5, amaximum stabilization effect is achieved starting from EDTA:Fe³⁺ molarratio of about 2:1. This corresponds to about 100 ppm EDTA at a Fe³⁺content of 10 ppm.

Example 11. Stability of OCT(C1) in Lipid Formulations with EDTA andIron in the Absence and Presence of ETA

Lipid formulations containing EDTA or EDTA(Na) in the absence andpresence of ETA were prepared according to the compositions given inTable 11. As shown in Example 1, neither EDTA(Na) nor EDTA are solublein EtOH/PG without using ETA. EDTA(Na) was also insoluble in EtOH/PGeven in the presence of ETA as assessed by visual inspection. Therefore,EDTA(Na), EDTA and EDTA(Na)/ETA containing mixtures in EtOH/PG wereadditionally filtered using a Millex-LG hydrophilic PTFE 0.2 μm syringefilter to remove the non-dissolved EDTA particles. After preparation,formulations were divided into sterilized 2R glass vials (0.5 g offormulation per vial), sealed and placed in a controlled environmentstorage cabinet at 40° C./75% RH. The headspace of the vials was ambientair to ensure forced degradation conditions, i.e., no inert atmospheresuch as nitrogen was introduced. At predefined sampling points (up totwo months of storage) two vials of each formulation and storagecondition were withdrawn from controlled environment cabinets,equilibrated to room temperature for 1 hour and analyzed for peptidecontent (assay) using gradient HPLC with UV detection.

TABLE 11 OCT(CI) containing lipid formulation compositions (in wt %)with different concentrations of Fe³⁺ and EDTA. The octreotide contentcorresponds to 20 mg/mL octreotide free base when corrected for peptidecontent, purity and formulation density. The SPC/GDO weight ratio was50/50 in all formulations. Sample ID OCT (Cl) SPC + GDO EtOH PG EDTAEDTA(Na) ETA FeCl₃ × 6H₂O** Sample 79 2.27 84.73 6.50 6.50 — — — —Sample 80* 2.27 84.72 6.50 6.50 — 0.01 — 0.00242 Sample 81* 2.27 84.716.50 6.50 — 0.01 0.00840 0.00242 Sample 82* 2.27 84.72 6.50 6.50 0.01 —— 0.00242 Sample 83 2.27 84.71 6.50 6.50 0.01 — 0.00840 0.00242 *Forpreparation of these formulations, EDTA mixtures in EtOH/PG werefiltered using Millex-LG hydrophilic PTFE 0.2 μm syringe filter toremove insoluble EDTA particles. **0.00242 wt % of FeCl₃ × 6H₂Ocorresponds to 5 ppm of Fe³⁺.

FIG. 6 presents the assay and Stability Index values of octreotide as afunction of time, respectively. As seen, only EDTA solubilized in thelipid formulation with the help of ETA dramatically enhanced the peptidestability compared to the reference formulation in the presence of 5 ppmFe³⁺. Under the same conditions, formulations containing EDTA(Na), EDTAor EDTA(Na)/ETA showed negative effect on OCT(C1) stability (vs. thereference formulation).

Example 12. Effect of Different Alkylamines and Solvents on Stability ofOCT(C1) in Lipid Formulations with EDTA

Lipid formulations were prepared according to the compositions given inTable 12. Formulations were divided into sterilized 2R glass vials (0.5g of formulation per vial), sealed and placed in a controlledenvironment storage cabinet at 40° C./75% RH. The headspace of the vialswas ambient air to ensure forced degradation conditions, i.e., no inertatmosphere such as nitrogen was introduced. At predefined samplingpoints (up to two months of storage) two vials of each formulation andstorage condition were withdrawn from the controlled environmentcabinets, equilibrated to room temperature for 1 hour and analyzed forpeptide content (assay) using gradient HPLC with UV detection.

TABLE 12 OCT(Cl) containing lipid formulation compositions (in wt %).The octreotide content corresponds to 20 mg/mL octreotide free base whencorrected for peptide content, purity and formulation density. TheSPC/GDO weight ratio was 50/50 and ETA:EDTA, DiETA:EDTA, andethylenediamine:EDTA molar ratios were 4:1 in all formulations. SampleID OCT (Cl) SPC + GDO EtOH PG EDTA ETA DiETA Ethylene diamine Sample 842.27 84.71160 6.50 6.50 0.01 0.0084 — — Sample 85 2.27 84.70560 6.506.50 0.01 — 0.01440 — Sample 86 2.27 84.71180 6.50 6.50 0.01 — — 0.00820

FIG. 7 presents the octreotide assay at different storage time points.As shown, the different alkylamines (ETA, DiETA or ethylenediamine) usedto solubilize 0.01 wt % (100 ppm) of EDTA into the lipid formulationsenhanced the peptide stability to a similar high degree when compared tothe reference formulation. The obtained results also show that thepositive effect of EDTA on the stability of OCT(CI) is independent onthe mixture used to prepare the lipid formulations as indicated by thedata for EtOH/PG containing formulations in FIG. 8 when compared withFIG. 9.

Example 13. Stability of SOM(C1) in Lipid Formulations in the Presenceof EDTA

Lipid formulations containing SOM(C1) in the absence and presence of 100ppm EDTA using EtOH/PG, were prepared according to the compositionsgiven in Table 13. Formulations were divided into sterilized 2R glassvials (0.5 g of formulation per vial), sealed and placed in controlledenvironment storage cabinets at either 40° C./75% RH or 25° C./60% RH.The head space of the vials was ambient air to ensure forced degradationconditions, i.e., no inert atmosphere such as nitrogen was introduced.At predefined sampling points (up to three months of storage) two vialsof each formulation and storage condition were withdrawn from thecontrolled environment cabinets, equilibrated to room temperature for 1hour and analyzed for peptide content (assay) using gradient HPLC withUV detection.

TABLE 13 SOM(Cl) containing lipid formulation compositions (in wt %)without and with EDTA. The SPC/GDO weight ratio was 50/50 in allformulations. Sample ID SOM(Cl) SPC + GDO EtOH PG EDTA ETA Sample 892.00 86.00000 10.00 2.00 — — Sample 90 2.00 85.98164 10.00 2.00 0.010000.00836

FIG. 9 presents the SOM assay at different storage time points andstorage conditions. As shown, the presence of 100 ppm of EDTAsolubilized in the lipid formulation by the use of ETA dramaticallyenhanced the peptide stability at both 40° C./75% RH and 25° C./60% RHstorage conditions.

Example 14. Stability of GOS(C1) in Lipid Formulations in the Presenceof EDTA and Iron

Lipid formulations containing GOS(C1) in the absence and presence of 100ppm EDTA were prepared according to the compositions given in Table 14.Formulations were divided into sterilized 2R glass vials (0.9 g offormulation per vial), sealed and placed in a controlled environmentstorage cabinet at 40° C./75% RH. The headspace of the vials was ambientair to ensure forced degradation conditions, i.e., no inert atmospheresuch as nitrogen was introduced. Both formulations also contained 5 ppmFe³⁺ to ensure additional oxidative stress conditions. At predefinedsampling points (up to two months of storage) two vials of eachformulation were withdrawn from controlled environment cabinets,equilibrated to room temperature for 1 hour and analyzed for peptidecontent (assay) using gradient HPLC with UV detection.

TABLE 14 GOS(Cl) containing lipid formulation compositions (in wt %)without and with EDTA. The SPC/GDO weight ratio is 50/50 in allformulations. Sample ID GOS(Cl) SPC + GDO EtOH DMSO EDTA ETA FeCl₃ ×6H₂O* Sample 93 1.00 78.99758 10.00 10.00 — — 0.00242 Sample 94 1.0078.97918 10.00 10.00 0.01 0.0084 0.00242 *0.00242 wt % of FeCl₃ × 6H₂Ocorresponds to 5 ppm of Fe³⁺

FIG. 10 presents the assay and Stability Index values of GOS as afunction of time, respectively. As evident, the use of 100 ppm EDTAsolubilized in the lipid formulation with the help of ETA significantlyenhanced the peptide stability in the presence of 5 ppm Fe³⁺. The dataindicate that EDTA provides protection of the peptide towards low tomoderate levels of metals that may originate from the excipients, theAPI or the processing equipment.

Example 15. Stability of OXY(C1) in Lipid Formulations in the Presenceof EDTA and Iron

Lipid formulations containing OXY(CI) in the absence and presence of 100ppm EDTA were prepared according to the compositions given in Table 15.Formulations were divided into sterilized 2R glass vials (0.9 g offormulation per vial), sealed and placed in a controlled environmentstorage cabinet at 40° C./75% RH. The headspace of the vials was ambientair to ensure forced degradation conditions, i.e., no inert atmospheresuch as nitrogen was introduced. Both formulations also contained 5 ppmFe³⁺ to enhance the oxidative stress conditions. At predefined samplingpoints (up to two months of storage) two vials of each formulation werewithdrawn from the controlled environment cabinets, equilibrated to roomtemperature for 1 hour and analyzed for peptide content (assay) usinggradient HPLC with UV detection.

TABLE 15 OXY(Cl) containing lipid formulation compositions (in wt %)without and with EDTA. The SPC/GDO weight ratio is 50/50 in allformulations. Sample ID OXY(Cl) SPC + GDO EtOH DMSO EDTA ETA FeCl₃ ×6H₂O* Sample 95 1.00 78.99758 10.00 10.00 — — 0.00242 Sample 96 1.0078.97918 10.00 10.00 0.01 0.0084 0.00242 *0.00242 wt % of FeCl₃ × 6H₂Ocorresponds to 5 ppm of Fe³⁺

FIG. 11 presents the assay and Stability Index values of O×Y as afunction of time, respectively. As evident, the use of 100 ppm EDTAsolubilized in the lipid formulation with the help of ETA significantlyenhanced the peptide stability in the presence of 5 ppm Fe³⁺. The dataindicate that EDTA provides protection of the peptide towards low tomoderate levels of metals that may originate from the excipients, theAPI or the processing equipment.

Example 16. Stability of GRN(0) in Lipid Formulations in the Presence ofEDTA and Iron

Lipid formulations containing GRN(0) in the absence and presence of 100ppm EDTA were prepared according to the compositions given in Table 16.Formulations were divided into sterilized 2R glass vials (1 g offormulation per vial), sealed and placed in a controlled environmentstorage cabinet at 40° C./75% RH. The headspace of the vials was ambientair to ensure forced degradation conditions, i.e., no inert atmospheresuch as nitrogen was introduced. Both formulations also contained 5 ppmFe³⁺ to ensure additional oxidative stress conditions. At predefinedsampling points (up to two months of storage) two vials of eachformulation were withdrawn from the controlled environment cabinets,equilibrated to room temperature for 1 hour and analyzed for peptidecontent (assay) using gradient HPLC with UV detection.

TABLE 16 GRN(0) containing lipid formulation compositions (in wt %)without and with EDTA. When corrected to assay and water content, theGRN concentration is 9.94 mg/g in all formulations. The SPC/GDO weightratio is 50/50 in all formulations. Sample ID GRN(0) SPC + GDO EtOH DMSOEDTA ETA FeCl₃ × 6H₂O* Sample 97 1.00 78.99758 5.00 15.00 — — 0.00242Sample 98 1.00 78.97918 5.00 15.00 0.01 0.0084 0.00242 *0.00242 wt % ofFeCl₃ × 6H₂O corresponds to 5 ppm of Fe³⁺

FIG. 12 presents assay and Stability Index values of GRN as a functionof time, respectively. As evident, 100 ppm EDTA solubilized in the lipidformulation with the help of ETA significantly enhanced the stability ofGRN in the presence of 5 ppm Fe³⁺. The data indicate that EDTA providesprotection of the active substance towards low to moderate levels ofmetals that may originate from the excipients, the API or the processingequipment.

Example 17. Stability of GOS(CI) in Phospholipid/Monoglyceride (SPC/GMO)and Phospholipid/Triglyceride (SPC/SbOil) Based Formulations in thePresence of EDTA and Iron

Lipid formulations containing GOS(CL) in the absence and presence of 100ppm EDTA were prepared according to the compositions given in Table 17.Formulations were divided into sterilized 2R glass vials (1 g offormulation per vial), sealed and placed in controlled environmentstorage cabinet at 40° C./75% RH. The headspace of the vials was ambientair to ensure forced degradation conditions, i.e., no inert atmospheresuch as nitrogen was introduced. All formulations also contained 5 ppmFe′ to enhance the oxidative stress conditions. At predefined samplingpoints (up to 9 weeks of storage) two vials of each formulation werewithdrawn from the controlled environment cabinets, equilibrated to roomtemperature for 1 hour and analyzed for peptide content (assay) usinggradient HPLC with UV detection.

TABLE 17 GOS(Cl) containing lipid formulation compositions (in wt %)without and with EDTA. The SPC/GMO and SPC/SbOil weight ratios were50/50 in all formulations. Sample ID GOS(Cl) SPC + GMO SPC + SbOil EtOHDMSO EDTA ETA FeCl₃ × 6H₂O* Sample 99 1.00 83.99758 — 10 5 — — 0.00242Sample 100 1.00 83.97918 — 10 5 0.01 0.0084 0.00242 Sample 101 1.00 —83.99758 10 5 — — 0.00242 Sample 102 1.00 — 83.97918 10 5 0.01 0.00840.00242 *0.00242 wt % of FeCl₃ × 6H₂O corresponds to 5 ppm of Fe³⁺

FIG. 13 and FIG. 14 present the assay and Stability Index values of GOSsolubilized in either SPC/GMO or SPC/SbOil based formulations as afunction of time, respectively. As shown, 100 ppm EDTA solubilized inboth formulation concepts with the help of ETA significantly enhancedthe peptide stability in the presence of 5 ppm Fe³⁺. The data indicatethat EDTA provides protection of the peptide towards low to moderatelevels of metals that may originate from the excipients, the API or theprocessing equipment.

Example 18. Lipid Oxidation in Placebo Lipid Formulations in thePresence of EDTA

Lipid placebo formulations in the absence and presence of 100 ppm EDTAwere prepared according to the compositions given in Table 18.Formulations were divided into sterilized 2R glass vials (1 g offormulation per vial), sealed and placed in controlled environmentstorage cabinets at either 60° C./ambient RH or 40° C./75% RH. Theheadspace of the vials was ambient air to ensure forced lipid oxidationconditions, i.e., no inert atmosphere such as nitrogen was introduced.Some formulations also contained 5 ppm Fe³⁺ to enhance the oxidativestress conditions (Table 18). At predefined sampling points (up to 9days of storage at 60° C./ambient RH and up to 30 days of storage at 40°C./75% RH) two vials of each formulation were withdrawn from thecontrolled environment cabinets, equilibrated to room temperature for 1hour and analyzed for oxygen concentration in the vial headspace (oxygenconsumption is here used as an indirect measure of lipid oxidation inthe lipid formulations) using a needle-type oxygen microsensor.

TABLE 18 Lipid formulation compositions (in wt %) without and with EDTA.The SPC/GDO weight ratio was 50/50 and 35/65 in Samples 103-106 andSamples 107-110, respectively. Sample ID SPC GDO EtOH EDTA ETA* FeCl₃ ×6H₂O** Sample 103 45.00 45.00 10 — — — Sample 104 45.00 45.00 10 — —0.00242 Sample 105 44.99 44.99 10 0.01 0.0116 — Sample 106 44.99 44.9910 0.01 0.0116 0.00242 Sample 107 31.50 58.50 10 — — — Sample 108 31.5058.50 10 — — 0.00242 Sample 109 31.49 58.49 10 0.01 0.0116 — Sample 11031.49 58.49 10 0.01 0.0116 0.00242 *ETA:EDTA molar ratio is 5.5:1**0.00242 wt % of FeCl₃ × 6H₂O corresponds to 5 ppm of Fe³⁺

The obtained results are summarized in FIG. 15, FIG. 16, FIG. 17 andFIG. 18. The data in the figures clearly show that, independent ofstorage condition, lipid ratio used to prepare formulations or thepresence of Fe³⁺, addition of 100 ppm of EDTA drastically reduceconsumption of oxygen (and thus lipid oxidation degradation) in allformulations. The data indicate that EDTA provides protection of thelipids towards low to moderate levels of metals that may originate fromthe excipients or the processing equipment.

Example 19. DTPA Solubility as a Function of ETA/DTPA Molar Ratio

0.08 wt % DTPA solutions in EtOH/PG (50/50 w/w) were prepared in theabsence and presence of various amounts of ETA added at differentETA/DTPA molar ratios (Table 19). The results show that DTPA is notsoluble in EtOH/PG without using ETA. The obtained results also showthat about 4.3 mol of ETA per 1 mol of DTPA is close to the requiredminimum amount needed to solubilize DTPA in the non-aqueous solventused.

TABLE 19 Solubility of 0.08 wt % DTPA in EtOH/PG as a function ofETA/DTPA molar ratio. ETA/DTPA DTPA solubility Sample ID (mol/mol) (ca24 h mixing) Sample 111 0.0 Not soluble Sample 112 1.7 Not solubleSample 113 4.3 Soluble Sample 114 4.8 Soluble Sample 115 6.2 SolubleSample 116 7.9 Soluble

The invention claimed is:
 1. A pre-formulation comprising: i) a lipidmixture comprising: a) glycerol dioleate (GDO); b) at least onephosphatidyl choline (PC); c) ethanol or mixtures of ethanol andpropylene glycol; and ii) ethylenediaminetetraacetic acid (EDTA) andethanolamine (ETA); wherein the pre-formulation has a water content of 0to 1.0 wt %, wherein the molar amount of ETA relative to the molaramount of EDTA is at least 3.5 (mol/mol).
 2. A pre-formulation of claim1 wherein the EDTA and ETA form an alkylammonium EDTA salt.
 3. Apre-formulation of claim 1 wherein the EDTA is present in an amount of0.001 to 0.05 wt % of the pre-formulation.
 4. A pre-formulation of claim1 wherein component (ii) comprises an alkylammonium counterion havingonly one amino or alkylamino group and wherein the ratio of EDTA: thetotal of the alkylammonium counterion and any amine free base thereof inthe pre-formulation is 1:≥3.0.
 5. A pre-formulation of claim 1 whereincomponent (ii) comprises an alkylammonium counterion having two or moreamino and/or alkylamino groups, wherein the ratio of EDTA: the total ofthe alkylammonium counterion and any amine free base thereof in thepre-formulation is 1:≥2.0.
 6. A pre-formulation of claim 1 furthercomprising an active agent (d).
 7. A pre-formulation of claim 6 whereinthe active agent (d) comprises a peptide, protein and nucleic acid basedactive agents, and hormones.
 8. A pre-formulation of claim 6 wherein theactive agent (d) comprises endogenous GLP-1, or a GLP-1 receptoragonist, or a salt thereof.
 9. A pre-formulation of claim 1 wherein thepre-formulation forms, or is capable of forming, at least one liquidphase structure upon contact with excess aqueous fluid.
 10. A medicamentcomprising the pre-formulation of claim
 1. 11. A pre-filledadministration device containing a pre-formulation of claim
 1. 12. A kitcomprising an administration device of claim
 11. 13. A method ofreducing oxidation of at least one active agent in a pre- formulationcomprising: i) a lipid mixture comprising: a) glycerol dioleate (GDO);b) at least one phosphatidyl choline (PC); c) ethanol or mixtures ofethanol and propylene glycol; and d) at least one active agent; and ii)ethylenediaminetetraacetic acid (EDTA) and ethanolamine (ETA); whereinthe pre-formulation has a water content in the range of 0 to 1.0 wt%;wherein the molar amount of ETA relative to the molar amount of EDTA isat least 3.5 (mol/mol).
 14. A process for preparing a pre-formulation ofclaim 1: dispersing EDTA and ethanolamine in ethanol or mixtures ofethanol and propylene glycol to produce a dispersion; mixing thedispersion until the EDTA and ethanolamine are fully dissolved toproduce a mixture; and adding glycerol dioleate (GDO), at least onephosphatidyl choline (PC), and at least one active agent to the mixtureto produce the pre-formulation.
 15. A pre-formulation of claim 1 whereincomponent a) is present in an amount of 20-90 wt. % of thepre-formulation.
 16. A pre-formulation of claim 1, wherein component b)comprises soy phosphatidyl choline (SPC).
 17. A pre-formulation of claim1 wherein component b) is present in an amount of 20-80 wt. % of thepre-formulation.
 18. A pre-formulation of claim 1 wherein component c)is present in an amount of 1-30 wt. % of the pre-formulation.
 19. Apre-formulation of claim 1, wherein component a) is present in an amountof 20-90 wt. %, component b) is present in an amount of 20-80 wt. %, andcomponent c) is present in an amount of 1-30 wt. %, based on the totalweight of the preformulation.
 20. A pre-formulation of claim 1, whereincomponent a) is present in an amount of 30-70 wt. %, component b) ispresent in an amount of 30-70 wt. %, and component c) is present in anamount of 2-20 wt. %, based on the total weight of the preformulation.21. A pre-formulation of claim 1, wherein component a) is present in anamount of 43-60 wt. %, component b) is present in an amount of 33-55 wt.%, and component c) is present in an amount of 2-15 wt. %, based on thetotal weight of the preformulation.
 22. A pre-formulation of claim 1,wherein the ratio of component a): component b) is 40:60 to 70:30.
 23. Apre-formulation of claim 1, wherein the molar amount of ETA relative tothe molar amount of EDTA is in the range of 3.5 to 10 (mol/mol).
 24. Apre-formulation of claim 1, wherein the molar amount of ETA relative tothe molar amount of EDTA is in the range of 3.5 to 7 (mol/mol).